Compositions and methods for bone formation and remodeling

ABSTRACT

The mechanism by which the high bone mass (HBM) mutation (G171V) of the Wnt coreceptor LRP5 regulates the canonical Wnt signaling was investigated. The mutation was previously shown to reduce Dkk protein-1-mediated antagonism, suggesting that the first YWTD repeat domain where G171 is located may be responsible for Dkk protein-mediated antagonism. However, we found that the third YWTD repeat, but not the first repeat domain, is required for DKK1-mediated antagonism. Instead, we found that the G171V mutation disrupted the interaction of LRP5 with Mesd, a chaperon protein for LRP5/6 that is required for the coreceptors&#39; transport to cell surfaces, resulting in less LRP5 molecules on the cell surface. Although the reduction in the level of cell surface LRP5 molecules led to a reduction in Wnt signaling in a paracrine paradigm, the mutation did not appear to affect the activity of coexpressed Wnt in an autocrine paradigm. Together with the observation that osteoblast cells produce autocrine canonical Wnt, Wnt7b, and that osteocytes produce paracrine Dkk1, we believe that the G171V mutation may cause an increase in Wnt activity in osteoblasts by reducing the number of targets for paracrine Dkk1 to antagonize without affecting the activity of autocrine Wnt.

REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation-in-part of application Ser. No.11/097,518, filed Apr. 1, 2005, which is a continuation-in-part ofapplication Ser. No. 11/084,668, filed Mar. 18, 2005, which iscontinuation-in-part of application Ser. No. 10/849,067, filed May 19,2004, which claims the benefit of provisional Application No.60/504,860, filed Sep. 22, 2003, the contents of all of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of therapeutic methods,compositions and uses thereof, in the treatment of bone fractures, bonedisease, bone injury, bone abnormality, tumors, growths or viralinfections as well as for modulating pathophysiological processesincluding but not limited to glucose metabolism, lipid metabolism,triglyceride metabolism, adipogenesis, tumorigenesis, neurogenesis andbone-related activity. More particularly, the methods and compositionsof the invention are directed to the stimulation, enhancement andinhibition of bone formation or bone remodeling.

BACKGROUND OF THE INVENTION

Osteoporosis is a major public health problem, and it is especiallyprevalent in aging populations (1, 15, 21). The majority of fracturesthat occur in people over the age of 65 are due to osteoporosis (15,40). Peak bone mass is a determining factor in establishing the risk ofosteoporotic fracture (Heaney et al., 2000), and studies indicate thatgenetic factors contribute significantly to the variance in peak bonemass. One of the genes that regulate bone mass has recently beenidentified via positional cloning. Loss of function mutations in lowdensity lipoprotein receptor-related protein 5 (LRP5), a co-receptor forthe canonical Wnt signaling pathway (27), were found to be associatedwith Osteoporosis-Pseudoglioma Syndrome (OPPG), an autosomal recessivedisorder which shows a reduction of bone density in humans (9). Inaddition, two independent kindreds that manifest familial High Bone Mass(HBM) phenotypes were found to harbor a Gly171 to Val substitutionmutation (G171V) in LRP5 (5, 22). More recently, additional HBMmutations were reported in the same structural domain of the G171Vmutation (36). Moreover, mice in which the LRP5 genes were inactivatedby gene targeting showed phenotypes similar to those of OPPG patients(16), and transgenic expression of LRP5G171V in mice resulted in HBM(2). Furthermore, mouse primary osteoblasts showed reducedresponsiveness to Wnt in the absence of LRP5 (16), and Wnt (9) oractivated beta-catenin (4) stimulated the canonical Wnt signalingactivity and induced the production of the osteoblast marker alkalinephosphatase (AP) in osteoblast-like cells. Together, these pieces ofevidence indicate that the canonical Wnt signaling pathway plays animportant role in the regulation of bone development.

Wnt

The Wnt family of secretory glycoproteins is one of the major familiesof developmentally important signaling molecules and has been shown toregulate a wide range of biological and pathophysiological processesthat include glucose metabolism, bone remodeling, adipogenesis,neurogenesis, stem cell biology, and tumorigenesis. The canonical Wntsignaling pathway is initiated by the binding of canonical Wnts to theirreceptor complexes consisting of LDL receptor-related protein (LRP) 5/6and frizzled (Fz) proteins. Through yet to be fully characterizedmechanisms, beta-catenin, which is degraded via ubiquitin-mediatedproteolysis in the absence of Wnts, is stabilized, leading to anincrease in the cytosolic level of β-catenin. Free beta-catenin entersthe nucleus and activates gene transcription in a complex with theTCF/LEF-1 transcription factors (61-66). In addition, the Wnt pathway isnegatively regulated by many naturally occurring antagonists includingthe Dickkopf (Dkk) family of polypeptides (67, 68). Dkk binds to LRP5/6and presumably leads to the inactivation of the receptor proteins (34).Both human and mouse genetic evidence indicates that the Wnt coreceptorLRP5 has an important role in the regulation of bone remodeling;hypomorphic or null alleles lead to early onset of osteoporosis (14),whereas different mutant alleles are associated with high bone massphenotypes (32, 5, 51). It has been previously shown that that themutation indirectly reduced Dkk-mediated antagonism of canonical Wntsignaling (69), thus suggesting the Dkk-LRP5 interaction as a potentialtherapeutic target for increasing bone mass.

Until recently, the canonical Wnt signaling pathway was believed tostart when Wnt bound to frizzled Fz proteins. The seven transmembranedomain-containing Fz proteins suppress the Glycogen synthase kinase 3(GSK3)-dependent phosphorylation of beta-catenin through ill-definedmechanisms involving Dishevelled proteins. This suppression leads to thestabilization of beta-catenin. Beta-catenin can then interact withtranscription regulators, including lymphoid enhancing factor-1 (LEF-1)and T cell factors (TCF), to activate gene transcription (7, 10, 38).Recently, genetic and biochemical studies have provided solid evidenceto indicate that co-receptors are required for canonical Wnt signalingin addition to Fz proteins (27, 28). The fly ortholog of LRP5/6 (LRP5 orLRP6), Arrow, was found to be required for the signaling of Wg, the flyortholog of Wnt-1 (37). LRP5 and LRP6 are close homologues whichbasically function the same way, yet exhibit, different expressionpatterns. In addition, LRP6 was found to bind to Wnt1 and regulateWnt-induced developmental processes in Xenopus embryos (34). Moreover,mice lacking LRP6 exhibited developmental defects that are similar tothose caused by deficiencies in various Wnt proteins (30). Furthermore,LRP5, LRP6 and Arrow were found to be involved in transducing thecanonical Wnt signals by binding Axin and leading to Axin degradationand beta-catenin stabilization (25, 35). The LRP5/6-mediated signalingprocess does not appear to depend on Dishevelled proteins (18, 31).Recently, a chaperon protein, Mesd, was identified as required forLRP5/6 transport to the cell surface (6, 11).

The involvement of the Wnt pathway in inducing repression or expansionof bone growth was demonstrated in a number of publications thatdescribed the various effects of mutations in LRP5 upon skeletalstructures that served to give rise to low bone mass (14, 88) orincreased bone mass (32, 5, 51). There is even a recently describedgenetically modified mouse model for osteoporosis, where disruption inboth chromosomal copies of LRP5 (a LRP5−/− knockout) generates a lowbone mass phenotype (89). However, it should be noted that even thoughthe above mentioned references are in regard to LRP5, it should beobvious that intervention in other points along the Wnt signalingpathway could also benefit from administration of compound that havebeen identified through the processes of the present invention. Forrecent reviews of the interconnections between the Wnt pathway and bonegrowth, (see cited references 82, 90, and 91).

Dkk Proteins

Xenopus Dickkopf (Dkk)-1 was initially discovered as a Wnt antagonistthat plays an important role in head formation (8). Thus far, fourmembers of Dkk have been identified in mammals (17, 26). These includeDkk1, Dkk2, Dkk3 and Dkk4. Dkk1 and Dkk2 inhibit canonical Wnt signalingby simultaneously binding to LRP5 or LRP6 and a single transmembraneprotein Kremen (3, 23, 24, 32). It has been previously reported that theLRP5 HBM G171V mutation appeared to attenuate Dkk1-mediated antagonismto the canonical Wnt signaling (5). The present invention describes themechanism for this attenuation.

The third YWTD repeat domain of LRP5 which is required for Dkk-mediatedantagonism of Wnt signaling has previously been identified (69). Inaddition, the Dkk-binding cavity and key residues within the cavity hasbeen delineated by site-directed mutagenesis (69). This cavity islocated at the large opening of the barrel-like structure of the YWTDrepeat domain that is made of six beta-propellers (FIG. 17A).Importantly, the two most important residues in the interaction withDkk, Residues Glu721 and Trp780 (69), are located at the bottom of thiscavity, suggesting that small molecule chemicals that bind to thiscavity may be able to disrupt the Dkk-LRP5 interaction by blocking theaccess to this key residue.

SUMMARY OF THE INVENTION

As described herein, the invention provides a model which explains thefunctional interactions of cavities on domains of receptors orco-receptors involved in bone formation or bone remodeling with Dkk,Wnt, Mesd, or other proteins which function in similar ways. Thesereceptors include, but are not limited to, the LRP5 receptor, the LRP6receptor, and the frizzled receptor. The LRP5 and LRP6 receptor containsfour YWTD repeat domains. Each domain contains multiple YWTD repeats ofamino acids. The LRP5 and LRP6 receptor also have an LDL receptorrepeat. Both LRP5 and LRP6 are close homologues and function inbasically the same way although they possess different expressionpatterns.

The invention provides methods for identifying non-native or exogenouscompounds which bind to or interact with these cavities to cause thestimulation, inhibition or regulation of Wnt signaling, and thus boneformation, tumorigenesis and any other biological and pathologicalprocess regulated by Wnt signaling. In particular, the invention isdirected to disheveled, beta-catenin or LEF-1/TCF protein or receptor.In a particular embodiment, the invention is directed to a method foridentifying a compound that interferes with the binding of protein toLRP5, LRP6, Wnt, Dkk, frizzled, disheveled, beta-catenin or LEF-1/TCFprotein or receptor. In one embodiment, the method comprises:

(a) identifying a compound that binds to LRP5, LRP6, Wnt, Dkk, frizzled,disheveled, beta-catenin or LEF-1/TCF protein; and

(b) determining if the compound identified in (a) modulates the bindingof a protein to LRP5, LRP6, Wnt, Dkk, frizzled, disheveled, beta-cateninor LEF-1/TCF protein or receptor.

The compounds in step (a) may be identified by:

(a) screening for a compound that fits into the cavity or binding siteof LRP5, LRP6, Wnt, Dkk, frizzled, disheveled, beta-catenin, orLEF-1/TCF using the UNITY program;

(b) docking said compound into the cavity using the Flexx program; and

(c) identifying the compound with the highest binding affinity using theCscore program.

In another embodiment, the method comprises:

(a) identifying a compound that modulates Wnt signaling; and

(b) determining whether the compounds identified in (a) interact or bindto LRP5, LRP6, Wnt, Dkk, frizzled, disheveled, beta-catenin or LEF-1/TCFprotein or receptor.

The compound identified may be a small molecule, protein peptide,polypeptide, cyclic molecule, heterocyclic organic molecule, nucleicacid, lipid, charged lipid, polar lipid, non-polar lipid, sugar,glycoprotein, glycolipid, lipoprotein, chemical, or a fragment of acompound that comprises a heterocyclic, organic molecule, nucleic acid,lipid, charged lipid, polar lipid, non-polar lipid, sugar, glycoprotein,glycolipid, lipoprotein, or chemical.

A non-native compound identified using the method of the presentinvention comprises a compound that is not naturally or normally foundin a cell or organism, as opposed to a native compound which is notintroduced from an outside source. As will be described in furtherdetail below, the compounds may be identified from a National CancerInstitute (NCI) database through various screening methods and assays.These compounds could also be modified to create derivates or analoguesnot found in the NCI database or in nature which also functioneffectively. Compounds are identified which disrupted Dkk and LRP5/6interactions, Wnt and LRP5/6 interactions and Mesd and LRP5/6interactions.

In a particular embodiment, the invention is directed to methods ofand/or compositions for modulating a pathophysiological process whichincludes but is not limited to glucose metabolism, cholesterolmetabolism, adipogenesis, tumorigenesis, neurogenesis and/orbone-related activity, as well as methods of or compositions fortreating a bone fracture, bone disease, bone injury or bone abnormality,diabetes, hyperglycemia or any metabolic disease using the compoundsidentified using the screening methods of the present invention. Inparticular embodiments, the compound may have the structure (I):

wherein at least one of R¹, R³, R⁴, R⁶, R⁸, R¹¹, R¹² or R¹³ is ahydrogen atom and wherein at least one of R¹, R³, R⁴, R⁶, R⁸, R¹¹, R¹²or R¹³ comprises an atom other than a hydrogen atom;the structure (II):

wherein at least one of R¹, R³, R⁴, R⁶, R⁸, R¹¹, R¹², R¹³ or R¹⁴ is ahydrogen atom and wherein at least one of R¹, R³, R⁴, R⁶, R⁸, R¹¹, R¹²,R¹³ or R¹⁴ comprises an atom other than a hydrogen atom;the structure (III):

wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷ or R⁸ is a hydrogenatom and wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷ or R⁸comprises an atom other than a hydrogen atom;the structure (IV):

wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ is ahydrogen atom and wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷ orR⁸, R⁹ or R¹⁰ comprises an atom other than a hydrogen atom; orthe structure (V):

wherein at least one of R¹, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³or R¹⁴ is a hydrogen atom and wherein at least one of R¹, R⁴, R⁵, R⁶,R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ or R¹⁴ comprises an atom other than ahydrogen atom.

These compounds are administered in the methods of the present inventionor are present in the compositions of the present invention in amountseffective to modulate said pathophysiological processes or to treat saidbone fracture, bone disease, bone injury or bone abnormality, diabetesor hyperglycemia to a subject in need thereof. In one embodiment, thesubject is a mammalian subject; in a particular embodiment, the subjectis a human subject.

In a specific embodiment, the invention is directed to isolatedcompounds having the following structures:

structure (VI):

wherein R¹⁵ is a linear or branched alkyl group;structure (VII):

wherein R¹³ and R¹⁴ are each independently H, or a linear or branchedalkyl group; orstructure (VIII):

wherein R¹³ is a linear or branched alkyl group or substituted orunsubstituted cycloalkyl group.

The invention is further directed to methods for obtaining compounds(VI)-(VIII). In a particular embodiment, compound (VI) is obtained byreacting gallocyanine with an alkyl halide under conditions promotingformation of said compound. Compound (VII) may be obtained by (a)reacting gallocyanine with an agent to replace the COOH group with aleaving group to obtain an intermediate; and (b) reacting the compoundobtained in step (a) with an alkyl amine to obtain said compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of wild-type LRP5 and itsdeletion mutants.

FIG. 2 illustrates that the G171V mutation disrupts LRP5 trafficking.HEK cells were transfected with expression plasmids as indicated in thefigure. One day later, the cells were lysed and immunoprecipitation wascarried out using an anti-Flag antibody. Mesd was Flag-tagged whereasall LRP5 molecules were HA-tagged. The G171V mutation disrupted theinteractions of both LRP5 with Mesd (FIG. 2A, lanes 1 and 3), and R12with Mesd (FIG. 2B, lanes 1 and 2), while the E721 mutation did notaffect the interaction (FIG. 2A, Lanes 2 and 3). The lower panels ofFIG. 2A and FIG. 2B show equal amounts of Wt and mutant LRP5 input forthe immunoprecipitation. [HEK cells were transfected with the Mesdplasmid and the expression plasmids indicated in the figure.] R12TGV,R12T, R1-4 and R1-4GV (GV) are AP fusion proteins, which are LRP5mutants lacking transmembrane domains that may be secreted in thesupernatants of the cell cultures. One day later, conditioned medium(CM) was collected and centrifuged at a high speed. The supernatantswere immunoprecipitated by an anti-HA antibody (FIG. 2C) or used for anAP assay (FIG. 2D). Cells were also lysed in the SDS-PAGE sample bufferand analyzed by Western blotting (lower panels of FIG. 2C and FIG. 2D).The data shows that the G171V mutation inhibited the secretion of R12and R1-4. FIG. 2E confirms that the G171V mutation interferes with cellsurface transport of LRP5 through the use of a binding assay whichdetects LRP5 on the cell surface. The levels of cell surface LRP5molecules were detected by Western analysis using streptavidin-horseradish peroxidase (SA-HRP) after the cell surfaces were biotinylated andLRP5 molecules were precipitated with anti-HA antibody (FIG. 2E, upperpanel). The levels of LRP5 in the immunocomplexes are shown in the lowerpanel of FIG. 2E.

FIG. 3 shows that the HBM G171V mutation of LRP5 is less susceptible toDkk1-mediated inhibition of coexpressed Wnt activity. The left panel ofFIG. 3A shows that when HEK cells were transfected with plasmids asindicated together with LEF-1 luciferase reporter plasmids in thepresence or absence of Wnt1, the HBM G171V mutation did not lead to anincrease in LEF-1-dependent transcriptional activity compared to thewildtype (Wt) LRP5 (LRP5_(Wt)). The right panel of FIG. 3A showsexpression levels of LRP5, LRP5_(G171V), LRP6, and LRP6_(G158V) asdetermined by antibodies specific to the HA tag carried by LRP5 proteinsor anti-LRP6 antibodies. FIG. 3B shows that when HEK cells weretransfected with LEF-1 luciferase reporter plasmids, Wnt-1, Dkk1 andKremen in the presence of Wt or G171V LRP5 as indicated in the figure.LEF-1 reporters-indicated Wnt activity is significantly higher in HEKcells expressing LRP5_(G171V) than those expressing LRP5_(Wt) when Dkkis present. The protein expression levels of Dkk1, Kremen and LRP5 wereverified by Western blotting, as shown in FIG. 3C.

FIG. 4 illustrates that cells expressing LRP5G171 show less Dkk1 bindingsites than those expressing LRP5_(Wt) (FIG. 4A). FIG. 4B shows equalamounts of Wt and mutant LRP5 expression after transfection.

FIG. 5 shows that the second domain of LRP5 is required for Wntactivity. HEK cells were transfected with LEF activity reporter plasmidsand expression plasmids. One day later, LEF reporter activity wasmeasured, as previously described. The results in FIG. 5 show thatLRP5_(R494Q) and LRP5_(G479V) (LRP5 with point mutations in the seconddomain) may abolish Wnt signaling compared to LRP5_(Wt).

FIG. 6 illustrates that the third domain of LRP5 is required forDkk-mediated antagonism. FIG. 6A shows that the third YWTD repeat domainis required for Dkk-mediated inhibition. HEK cells were transfected withLEF activity reporter plasmids, Kremen1 plasmids and expressionplasmids. LRP5R12 or LRP5R124, but not LRP5R34, could still potentiateWnt-stimulated LEF-1 activity, suggesting that either LRP5R12 orLRP5R124 retains the Wnt coreceptor function. However, Dkk1 could notinhibit Wnt signaling when LRP5R12 or LRP5R124 was present despite thecoexpression of Kremen. This suggests that the third YWTD repeat domainis required for Dkk1-mediated inhibition. The expression level ofLRP5_(Wt) and its mutant molecules are shown in FIG. 6B. FIG. 6Cillustrates that LRP5R34 contains Dkk1 binding sites and that E721 inR34 is required for Dkk1 binding. FIG. 6D is a schematic representationof the mutations.

FIG. 7 shows that amino acid residues in the third YWTD repeat domain,consisting of interaction surfaces, are required for Dkk-mediatedinhibition of Wnt. In FIG. 7A, the space filled model of the third YWTDrepeat domain was deduced based on the structure of the LDL receptorYWTD repeat domain (13). Based on the three-dimensional structure, 19LRP5 mutants containing Ala substitution mutations on the surface of thethird YWTD repeat domain were generated. The ability of these mutantLRP5 proteins to resist Dkk1-mediated inhibition was determined. Nine ofthe mutants (more than 5%) showed altered sensitivity to Dkk1-mediatedinhibition, and they all contained mutations that were localized on thesame surface. In FIG. 7B, HEK cells were transfected with LEF activityreporter plasmids, Kremin1 plasmid, and expression plasmids. Theexpression of Wt and mutant LRP5 molecules are shown in the lower panel.Among 19 mutations, the E721 mutation showed the strongest effect onDkk1-mediated inhibition of Wnt, followed by W781, and then Y719.LRP5_(G171v) also showed an effect on the Dkk1-mediated inhibition ofWnt.

FIG. 8 shows the two dimensional structures of three compounds obtainedfrom the National Cancer Institute (NCI). NCI106164 (FIG. 8A) shows a68% inhibitory effect on Dkk1 binding while NCI39914 (FIG. 8B) andNCI660224 (FIG. 8C) increase Dkk1 binding by 654% and 276%,respectively.

FIG. 9 illustrates the two-dimensional structure of anthra-9,10-quinone(FIG. 9A), a common substructure in NCI39914 and NCI660224. FIG. 9Bshows the two-dimensional structure of NCI 657566. FIG. 9C shows thetemplate that was used for the two-dimensional similarity search.

FIG. 10 shows the two-dimensional structure of compounds NCI366218(IIC8, FIG. 10A) and NCI8642 (IIIC3, FIG. 10B) which specificallyinterrupt Dkk1-LRP5 interaction and reverse the inhibition of Wntsignaling by Dkk1.

FIG. 11 illustrates that NCI366218 and NCI8642 reverse Dkk1 inhibition.HEK cells were transfected with LRP5 plasmid together with a LEF-1expression plasmid, LEF-1 luciferase reporter plasmid and a GFPexpression plasmid. The cells were then treated with differentconcentrations of the NCI366218 and NCI8642 compounds and subsequentlytreated with control CM, Wnt3a CM or Wnt 3a/Dkk1 CM mixture for 6 hrs.The reporter activity from cells treated with DMSO was taken as 100%.FIG. 11 shows that at certain concentrations, NCI366218 (FIG. 11A) andNCI8642 (FIG. 11B) can significantly reverse Dkk-mediated inhibition ofWnt activity.

FIG. 12 shows that NCI366218 and NCI8642 can inhibit Dkk1 binding toLRP5. HEK cells were transfected with Mesd plasmids and LRP5 or LRP5R34.One day later, cells were treated with different concentrations ofNCI366218 and NCI8642 and incubated on ice with conditioned medium (CM)prepared from HEK cells expressing mDkk1-AP. The AP activity wasdetermined as previously described. The AP activity from cells treatedwith DMSO was taken as 100%. FIG. 12 shows that NCI366218 (FIG. 12A) andNCI8642 (FIG. 12B) inhibit Dkk1 binding to LRP5 wt, and Dkk proteinbinding to LRP5R34.

FIG. 13 illustrates that NCI366218 (IIC8) can stimulate osteoblastdifferentiation. Bone marrow stromal (BMS) cells were isolated from3-month-old mice carrying a Green Fluorescent Protein (GFP) transgenecontrolled by the 2.3 Kb CollA1 promoter (2.3Col-GFP)¹¹, in which GFPcan be used as a marker of osteoblast cells. On the 8^(th) and 12^(th)day, cultures were treated with 9 μM and 26 μM of IIC8 compound,respectively. The same time point, cultures were treated with DMSO asthe control. FIG. 13 shows that the osteoblast differentiation marker,2.3Col-GFP, was turned on when BMS culture was treated with IIC8.

FIG. 14 shows an osteogenic assay. Primary bone marrow stromalosteoblasts were cultured in the presence and absence of NCI366218 andinduced into differentiation. 20 days later, mineralization of theosteoblasts reflecting the bone formation process was observed withXylene Orange staining. NCI366218 stimulated mineralization two fold.

FIG. 15 illustrates that both LRP5R12 and LRP5R34 contain Dkk1 bindingsites, E721 in R34 is required for the Dkk1 binding and the G171V LRP5mutant can abolish the Dkk binding to the cell surface. FIG. 15A showsthat Dkk1 can bind to both LRP5R12 and LRP5R34, but the Dkk1 binding tothe cell surface was significantly low in cells transfected with R12GV(G171V mutation in LRP5R12) and R34E (LRP5R34 carrying the E721mutation). FIG. 15B shows equal amounts of Wt and mutant LRP5 expressionafter transfection.

FIG. 16 illustrates Dkk1 and Wnt7b expression in osteogenic cells. TotalRNA was isolated from bone marrow stromal cell culture at different timepoints after differentiation induction. Dkk and Wnt expression level wasdetermined by real time RT-PCR. Wnt7b showed marked increase in itsexpression after differentiation induction (FIG. 16A). The ability ofWnt 7b to stimulate the LEF-1 reporter gene was examined and it was ableto stimulate the canonical Wnt pathway (FIG. 16B). FIG. 16C is mouselong bone section in situ hybridization picture. It shows most of Dkk1is expressed in osteocyte. FIG. D illustrates the interactions betweenKremen, Dkk, LRP, Wnt and Fz.

FIG. 17 shows structures of Dkk-binding cavity and chemicals. FIG. 17Ashows the three key residues that were used in our initial shape-basedvirtual screen. The yellow box denotes the cavity. FIGS. 17B-G shows thechemical structures of IC13 (B), IC15 (C), IIC8 (D), IIC15 (E), IIC24(F) and IIIC3 (G). The anthra-9,10-quinone core is boxed.

FIG. 18 shows effects of compounds on Wnt activity and Dkk-binding. InFIGS. 18 A and B, cells were transfected with Wnt activity reportergene. Varying concentrations of compounds were added with Wnt3a CM,Wnt3a+Dkk1 CM, or control CM (Basal). Six hours later, Wnt reporter geneactivity was determined. (AU: arbitrary unit). The errors are less than5%. (C, D) Cells were transfected with wildtype LRP5 (C) or LRP5R34 (D).The binding of Dkk-1-AP to the cells was determined. The data with thebinding of Dkk-1-AP to cells expressing LacZ being subtracted arepresented.

FIG. 19 shows Wnt antagonistic compounds and molecular modeling. FIG.19A shows effects of IC15 on Wnt3a-stimulated Wnt reporter gene activityand Dkk1-AP binding to LRP5. FIGS. 19B-E show molecular modeling of thebinding of IIIC3 and IC 15 to the second and third YWTD repeat domainsof LRP5. Corresponding residues are in parentheses.

FIG. 20 shows the effects of IIIC3 on bone formation. FIGS. 20A and 20Bshow effects on calvarial bone formation. Control vehicle (a), b-FGF(b), or IIIC3 (c) was injected under the skull derma three times a dayfor five days. Two weeks after the final injection, calvarias werecollected, fixed, decalcified, and sectioned. The new bone layers aremarked. The thickness of the new bone layers was quantified and is shownin B. FIGS. 20C-E shows effects on bone mineral density (BMD). BMD andbody weight were determined after the mice (n=20) were subjected totreatment and rest regiments.

FIG. 21 shows the effects of EnzoM01 and IC15 on the inhibition ofWnt-stimulated beta-catenin activity.

FIG. 22 shows the inhibition of Wnt-stimulated beta-catenin activity byboth EnzoM14 and EnzoM15.

FIG. 23 shows the reversal of Dkk1 inhibition of Wnt signaling activityby EnzoM02 (FIG. 23A) and EnzoM03 (FIG. 23B).

FIG. 24 shows the effects of three compounds on the viability of PC-3tumor cells at different cell densities: (A) 500 cells/well, (B) 1000cells/well and (C) 2000 cells/well.

FIGS. 25A and B show the effects of EnzoM01 on beta-catenin productionin different tumor cell lines.

FIG. 26 shows the effect of EnzoM01 (FIG. 26A), IC15 (FIG. 26B) andIIIC3 (FIG. 26C) on glucose metabolism in mice subjected to a highcaloric diet.

FIG. 27 shows the effect of (FIG. 27A) EnzoM01 and (FIG. 27B) IC15 onserum levels of triglycerides and cholesterol in high caloric diet fedmice.

FIG. 28 shows the effect of IIIC3 on glucose (FIG. 28A) and insulin(FIG. 28B) levels in db/db mice.

FIG. 29 shows a comparison of amino acids at the binding sites at thesecond (red) and third (blue) domains. Common amino acids are in black.

DETAILED DESCRIPTION OF THE INVENTION

The terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. That the upper andlower limits of these smaller ranges may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

The present invention has identified compounds which, when provided to acell, bind to, interact with or fit into sites or cavities found on thedomains of the co-receptors involved in the stimulation, enhancement,inhibition or regulation of bone formation, or bone remodeling. Thesereceptors include the LRP5 receptor, the LRP6 receptor, the frizzledreceptor or any other receptor involved in the LRP5 or LRP6 (LRP5/6)receptor system: LRP5 and LRP6 are commonly referred to as LRP5/6 in theliterature due to a number of shared features with regard to how theyare involved in the canonical Wnt system: LRP5 and LRP6 share a 70%homology on the amino acid level. Due to this similarity, it is expectedthat many of the compounds that were selected for an ability to bind toLRP5 should also be able to interact with the LRP6 receptor as well. Thefrizzled receptor is a co-receptor that has a domain containing CRD, aWnt-binding site which functions to increase or decrease Wnt activity.

The LRP5 and LRP6 receptors contain YWTD repeat domains. In general, theareas comprising the YWTD repeat domains of the LRP5 or LRP6 receptorshare sufficient amino acid homology with each other that similarstructures are formed by each of these domains, but they are dissimilarenough that they vary in some of the dimensions of the pockets formedand have differences in some of the amino acids that are likely to beimportant in protein/protein interactions. In a particular embodiment,compounds used in the methods and/or compositions of the presentinvention interact with or bind to the 3^(rd) YWTD domain of the LRP5and/or LRP6 receptor. Such compounds may or not be able to bind to otherYWTD domains. In a particular embodiment, the compounds may bind to the1^(st) or 2^(nd) YWTD domain of the LRP5 and/or LRP6 receptor.

Some of these compounds may disrupt the Dkk and LRP5 interaction. Othercompounds inhibit Wnt signaling by inhibiting the binding of Wnt toLRP5/6. As will be described herein below, the invention has alsoidentified compounds that modulate interactions between Dkk and kremin,LEF/TCF-1 and beta-catenin, Wnt and frizzled. The compounds of thepresent invention are non-native or exogenous compounds which are notpresent in the cell, but originate from an outside source. They compriseagonists, which are agents that can combine with the receptors toinitiate events, antagonists, which are agents that combine with thereceptors to inhibit the effect of agonists, and partial agonists, whichhave characteristics of both agonists and antagonists—at times appearingto cause actions and at other times inhibiting actions by decreasing theeffects of agonists, for example. Some of these compounds may alsoincrease affinities, or the degree to which drugs or compounds areattracted to receptor binding sites.

Identification of Candidate Compounds

The compounds used in the compositions and methods of the presentinvention are identified using screening methods described herein.

Screening Compounds that Interact with the Specified Domains of LRP5

Screening Compounds Using Domain III of LRP5 as a Template

Virtual Screening

In a specific embodiment, the UNITY™ program (Tripos, Inc.) may be usedto screen the National Cancer Institute (NCI) database(http://129.43.27.140/ncidb2) for chemical compounds that are able tofit into the cavity on Domain III of the YWTD repeat domain of the LRP5receptor. This database is freely searchable and includes thecoordinates of 250, 251 small chemical compounds. A search query isdesigned to consist of R764 and E721 with 0.3 Å tolerance, and ahydrophobic center with 1.0 Å tolerance that is 3.2 Å away from Trp781,pointing towards the cavity. Taking the flexibility of the compoundsinto consideration, the Directed Tweak algorithm in the UNITY™ programallows for a rapid, conformationally flexible three dimensional search(21).

The candidate compounds obtained using the UNITY™ program are thendocked into the Dkk1 binding surface using the FlexX™ program (Tripos,Inc.) for energy minimization (17), which quickly and flexibly docksligands to protein-binding sites (44). Residues E721, W864, Y719, R764,D877, F888, G782, W781 and M891, shown to be critical for Dkk1recognition (FIG. 7A), are considered in the calculations. Following thedocking procedures, the compounds are then ranked based on theirpredicted ability to bind to the Dkk1 binding pocket using the Cscore™program. Cscore™ generates a relative consensus score based on how wellthe individual scoring functions of the protein-ligand complexesperformed (8). The Cscore™ is then subjected to final manual visualinspection.

Biological Assays

Dkk-1 Binding Assay

The compounds identified may be screened for their ability to affectbinding of Dkk-1 to LRP-5 by methods known in the art, e.g., by Dkk-APPassay (see Examples and (69)).

Wnt Activity

The compounds identified may also be screened for Wnt activity. Thesecond and third domains of LRP5 are required for Wnt signaling, andthese domains probably directly interact with Wnt molecules. Since thesedomains share extensive amino acid sequence homology, it is probablethat certain compounds that bind to the third domain may also bind tothe first two domains, potentially causing the inhibition of Wntactivity. The compounds may be examined for the following: 1) basalreporter activity inhibition; 2) Wnt activity inhibition; and 3)reversal of Dkk-mediated inhibition of Wnt activity.

Osteogenic Assays

Compounds identified may be tested using in vitro or in vivo osteogenicassays.

(a) In Vitro Assays

Wnt stimulates the proliferation and differentiation of culturedosteoblasts and Dkk inhibits this process. Therefore, these compoundsincrease osteogenesis. This may be monitored by the examination ofmineralization or the expression of osteogeneic markers, including theexpression of BSP, osteocalcin, and collagen.

(b) In Vivo Assays

Testing for the effectiveness of these compounds in vivo may beconducted to determine if the compounds increase osteogenesis in vivo. Avariety of compound doses may be injected at the outer surfaces ofcalvarias and into bone marrow cavities. Increased bone formation may beexamined histologically and through the use of pQCT, DNX, and X-rayradioautography.

Beta-Catenin Level Assay

Cytosolic B-catenin is stabilized by Wnt signaling. The effect of thesecompounds on Wnt signaling may be examined by the resulting levels ofβ-catenin.

Phosphorylation of PPPSP Sites of LRP5/6

It was recently discovered that Wnt stimulates the phosphorylation ofLRP5 at PPPSP motifs at the intracellular domain of LRP5 (49).Antibodies specific to phosphorylated PSPPP may be obtained and used toexamine Wnt activity as described in the examples and as known in theart (49).

Screening Compounds Using Domain II of LRP5 as a Template

Virtual Screening

The structure of this domain may be deduced using homology modeling, asdescribed above. As described in the Examples, site-directed mutagenesisis used to map the residues that are required for Wnt signaling. Virtualscreening methods are applied to this Wnt signaling surface using themethods described above. Since domain II is involved in Wnt signaling,compounds identified using domain II as a template may increase Wntsignaling or decrease Wnt signaling. Since domain II and domain III arehomologous, the compounds identified using virtual screening may: 1)increase Dkk binding; 2) decrease Dkk binding; 3) increase Dkkantagonism; and/or 4) decrease Dkk antagonism.

Biological Assays

Compounds are tested using biological assays described above. Compoundsthat increase or decrease Wnt activity are identified using methodsdescribed above. Compounds that enhance or inhibit Dkk1 binding aredetermined using assays described above. Compounds that enhance orinhibit Dkk1 antagonism are determined using assays described above.

Screening Compounds by Using Domain I of LRP5 as a Template

Virtual Screening

The structure of this domain may be deduced using homology modeling, asdescribed above. Site-directed mutagenesis is used to map the residuesthat are required for Mesd binding and function, as described in FIG. 2.Virtual screening methods are applied to this Mesd-binding surface usingthe methods described in Example 5.1(A). Since domain I is involved inMesd functions, compounds identified using domain I as a template mayincrease or decrease LRP5 presentation to the cell surface, therebyincreasing or decreasing Wnt signaling and/or increasing or decreasingDkk antagonism. Since domain I and domain II are homologous, thecompound identified using virtual screening may increase or decrease Wntsignaling. Since domain I and domain III are homologous, the compoundsidentified using virtual screening may: 1) increase Dkk binding; 2)decrease Dkk binding; 3) increase Dkk antagonism; and/or 4) decrease Dkkantagonism.

Biological Assays

Compounds that increase or decrease Wnt activity are identified usingmethods described above. Compounds that enhance or inhibit Dkk1 bindingand antagonism may be determined in a particular embodiment using assaysdescribed in Example 5.1. Compounds that affect Mesd function aredetermined using assays shown in FIG. 2.

Screening Compounds that Interact with Other Regions of LRP5

The three domains of the extracellular portion of LRP described hereinabove are not the only potential sites that are candidates for virtualscreening. For instance, the EGF repeats that are also in theextracellular portion of LRP 5 are likely to be binding sites forprotein/protein interactions and could be used with the methods of thepresent invention. Furthermore, the intracellular portion of LRP5 isknown to have sites that are involved in protein interactions that arealso potential target sites.

Screening of Compounds that Interact with the CRD of the FrizzledReceptor

Wnt signals through a transmembrane receptor of the frizzled family.This frizzled receptor passes through the cell membrane several times. Aconserved cysteine-rich domain (CRD) located on the N-terminalextracellular region of frizzled acts as a Wnt binding site. Secretedfrizzled-related protein Frzb-1 contains CRD and serves as an antagonistof Wnt signaling expression.

The crystal structures of the CRDs of Frizzled 8 and secretedFrizzled-related protein 3 from mice have been determined (12). The Wntbinding sites may be determined by Wnt-binding and mutagenesis assays.

Virtual Screening

Virtual screening methods described above are used to screen forpotential compounds that interact with CRD to regulate the Wnt signalingpathway. A homology model is created using the known CRD structure frommouse protein as a template. Homology models for other frizzled familymembers or for human frizzled protein CRD regions are created. Based onthe structure and the amino acids involved in the CRD-Wnt interaction,energy minimization methods were used to screen for compounds to furthertest the biological activity of each compound. For those that showedhigher biological activity, a similar structural query was used toidentify additional candidate compounds.

Biological Assays

Wnt-binding assays may be used to screen the effect of compounds on theCRD region of frizzled proteins. CRD peptides (or frizzled proteins)expressed on the surface of the cell with a detectable marker (e.g,Myc-tag). Medium containing the compound and Wnt-alkaline phosphatasefusion protein (e.g Wnt8-AP) was used. After incubation, binding wasdetermined using immuno-histochemistry staining.

Once the candidate compounds show an effect on Wnt binding, otherbiological assays (as described above are applied to determine eachcompounds effect on Wnt signaling. (27, 38, 12)

Screening of Compounds that Interact with LRP6

LRP5 and LRP6 are commonly referred to as LRP5/6 in the literature dueto a number of shared features with regard to how they are involved inthe canonical Wnt system. Therefore, many of the compounds selected forthe ability to bind to LRP5 should also be able to interact with LRP6 aswell. However, LRP5 and LRP6 just share 70% homology on the amino acidlevel. Therefore the sequence of LRP6 may be used as a template toidentify effectors of the Wnt system in a similar manner to the way thatLRP5 to obtain compounds used in the method of the present invention.Each of the first, second and third YWTD repeat domains of LRP6 can beused as selective sites for predicting interactions of a virtual libraryof chemical compounds. In turn, it is expected that many of thecompounds selected for binding to LRP6 could also interact with LRP5.

Screening of Compounds that Interact with Dkk

Virtual Screening

The structure of Dkk1 is solved and its interaction surfaces to Kremenand LRP5/6 may be mapped using mutagenesis, as described in the examplesherein. Virtual screening is conducted according to the methodsdescribed above. Compounds are found to increase or decrease Dkk bindingto LRP5 or Kremen, or increase or decrease Dkk-mediated inhibition ofWnt.

Biological Assays

Compounds that increase or decrease Dkk binding to LRP5 are determinedas described above. Compounds that increase or decrease Dkk binding toKremen are determined as described above with the exception that thecells are transfected with Kremen instead of LRP5. Compounds thatincrease or decrease Dkk antagonism may in a particular embodiment bedetermined as described in Example 5.1.

Screening of Compounds that Interact with Dishevelled (DSL) Domains

Cytoplasm disheveled (DSL) proteins are activated by the Wnt-frizzledreceptor complex. They are essential in both canonical and non-canonicalWnt signaling pathways. DSL proteins are composed of an N-terminal DIXdomain, a central PDZ domain, and a C-terminal DEP domain. These threeconserved domains each associate with different proteins, thereby eachfunctioning in a different pathway.

The DIX domain exists as a homodyne and forms a predominantly helicalstructure. This was determined using pulsed-field gradient NMR studies.The DIX domain mediates targeting to acting stress fibers and cytoplasmvesicles in vivo. It thereby may represent a point of divergence in Wntsignaling. The stabilization of β-catenin through canonical Wntsignaling involves membrane targeting of DSL. Lees 58, Ser 59 and Met 60in mouse Dv12 are critically involved in the acting interaction. Lees 68and Glue 69 are important in cytoplasm vesicle localization.

The PDZ domain interacts with several molecules and plays an importantrole in both the canonical and non-canonical Wnt pathways. The threedimensional Xenopus PDZ domain structure has been determined (7).Through the use of chemical-shift perturbation NMR spectroscopy andbinding assays, there is a direct interaction between the conservedmotif KTXXXW of frizzled and the PDZ domain of mouse Dvl1. This allowsthe binding region to be determined (57).

The DEP domain of DSL proteins transducers signals to effector proteinsdownstream of Dvl in the Wnt pathway. The DEP domain of disheveled isrequired for the upregulation of β-catenin activity and the stimulationof Lef-1 mediated transcription in mammalian cells. The mouse Dvl1 DEPdomain's structure has been determined. (Wong, et al) It has been shownthat Lys434, Asp445, and Asp 448 play an important role inprotein-protein interaction, and that their mutations Wnt-1 inducedLef-1 activation.

Virtual Screening

Since the functional residues and secondary structures of the DIX domainhave been determined, a screening of the existing protein domains mayprovide information for tertiary structural configurations and potentialcandidates, and a simulation for the same may generate candidatecompounds for binding analysis. Candidate compounds affecting bindingmay be analyzed, and a new group of similar compounds may be assayedbiologically.

Since the three dimensional structure for PDZ and DEP is known, avirtual screening method similar to the method described above may beused. This structure may be used as a template to create a homologymodel for human protein domains or other similar functional proteindomains. Based on the structure and the amino acids involved inspecified functions, energy minimization methods may be used to screencompounds. The biological activity of each compound may be tested. Forthose compounds that show high biological activity, a similar structuralquery may be used to find more candidate compounds, and biologicalactivity will be further assayed.

Biological Assays

Actin-binding inhibition assays for actin binding regions, and Xnr3 orSiamois expression levels may be used for DIX domain vesiclelocalization. A constructed vector containing tagged DIX may betransfected into a cell, after compound treatment. Immunofluorescencestaining may then be used to determine actin-binding inhibition. RT-PCRmay be used to detect Xnr3 or Siamois levels for vesicle localization.

An in vitro binding assay may be used for initial screening for the PDZdomain. A peptide (e.g, Drp C terminal region) that binds to the PDZdomain of Dvl may be used. Purified tagged peptide bound to beads may bemixed with the PDZ domain and each compound, and after incubation,antibody may be used to detect the bound compounds. The bindingefficiency effect of each compound may be determined.

To screen for compounds that will affect the canonical Wnt pathway, aluciferase assay may be used for the domain. Cells may be transfectedwith the Dvl domain. Once these cells are incubated with compounds,Wnt/β-catenin activated luciferase activity may be assayed, therebymeasuring each compound's effect.

The compounds are then classified based on their structure, and theidentified compounds are further screened. Once candidate compoundsaffecting protein binding are identified, other biological assaysdescribed above may be used to determine the effect of each compound onWnt signaling (57, 6, 58, 55, and 7).

Screening of Compounds that Interact with β-Catenin

β-catenin mediates the transmission of the Wnt signal into the nucleusand thereby activates the target genes. The Wnt signal preventsβ-catenin degradation, allowing β-catenin to accumulate and subsequentlytranslocate to the nucleus to form a transcriptional activating complexwith members of the Tcf/LEF family of proteins.

The crystal structure of β-catenin, as well as the complex it forms withAxin, Lef, TCF and several other proteins, have been solved. Thisinformation may be used for the screening of compounds that regulatecanonical Wnt signaling.

β-catenin contains N-terminal armadillo repeats, which are the bindingsites for APC, LEF/TCF, E-cadherin and conductin/axin. All the bindingsites are located in armadillo repeat units 3-8 of β-catenin. Thebinding of the factors occupy the groove and thus preclude thesimultaneous binding of other competing β-catenin partners.

Virtual Screening

A modified strategy similar to the virtual screening described above maybe used for identifying compounds for β-catenin interaction for binding.The homology model of β-catenin from different species may be generatedusing β-catenin as a template. Based on the structure and the criticalamino acids involved in the interactions with LEF/TCF, Axin and APC,energy minimization methods may be used to screen for compounds tocreate groups of candidate compounds. Since all the aforementionedproteins occupy similar positions on β-catenin, when biological assaysare used for the screening of each compound, all four interactions aretested. Based on initial biological activity, the structures ofeffective compounds are analyzed, and new groups of compounds are testedusing similar methods. Additional biological assays may be carried outto identify the most effective compounds.

Biological Assays

Since all the β-catenin partners occupy similar positions, in vitrotranslation and protein binding assays may be used to determine theeffectiveness of each compound. Tagged β-catenin, TCF, APC, LEF or Axinconstructs may be transcribed and translated in vitro. Once they areincubated with the compounds, immunoblotting may be used to detectbinding.

Once compounds are identified which affect Wnt binding, other biologicalassays may be used, as described in section 5.1(B), to determine theeffect of each compound on Wnt signaling (52, 43, 16, 59, and 11).

Screening of Compounds that Interact with LEF-1/TCF TranscriptionFactors

Lymphoid enhancer-binding factor (LEF) is a DNA-binding protein thatplays an architectural role in the assembly and function of a regulatorynucleoprotein complex. It recognizes specific nucleotide sequencesthrough a high-mobility-group (HMG) domain. The solution structure ofthe HMG domain of mouse LEF-1, complexed with a 15-base-pairoligonucleotide duplex containing the optimal binding site from theTCR-alpha gene enhancer, has been solved.

Virtual Screening

A strategy similar to the virtual screening described above may be usedto screen for potential compounds that interact with HMG-oligonucleotidebinding, to thereby affect the activity of gene expression regulation.Based on the structure, proteins containing HMG domains bend the DNA towhich they bind. Any compounds that affect DNA bending or bindingability have an effect on the regulation of gene expression. Thehomology model for the LEF HMG domain for different species may becreated using the known structure as a template. Based on the structureand the amino acids involved in HMG-oligo interaction, energyminimization methods may be used to screen for compounds. Compoundswhich may either force the bending or prohibit the bending are selected.The DNA binding activity is used to screen the compounds. For compoundswhich show a much higher or much lower biological activity, a similarstructural query may be used to identify additional candidate compounds.

Biological Assays

DNA-binding assays may be used to screen the compounds. Oligonucleotidesand HMG domains are incubated with the compounds. Gel retardation assaysare used to determine the DNA binding. The binding experiment may bemodified with uniformly ¹³C labeled NMR to analyze domain bending. SinceLEF controlled gene regulation is directly affected, a luciferase assaymay also be used for detecting compound effects. Once compoundsaffecting protein binding are identified, other biological assays may beused to determine the effect of each compound on Wnt signaling (33).

In carrying out the virtual screening of a library it is understood thatthe library itself can be a physical library (as exemplified byscreening of the NCI library) or it can be a virtual library (asexemplified in Example 6 with compounds Enz 1-Enz 72). The library canbe made up of any compounds that can interact with the protein target ofinterest and affect its interaction with another protein. Examples ofsuch compounds can include but not be limited to organic molecules,peptides, and nucleic acids. The peptides can include but not be limitedto a library of peptides of random nature, a permutational series ofamino acids, fragments of antibodies to the protein of interest andfragments of a protein that interacts with the protein of interest. Thenucleic acids can include but not be limited to aptamers and a libraryof protein binding sequences.

The Use of Structural Comparison to Find More Effective Compounds

Knowledge of the effectiveness of a compound also allows the purposefuldesign of a family of novel analogues or compounds that have variationsin the groups that are appended to a core structure of the initialcompound. The novel compounds obtained in a particular embodimentmodulate the binding of a protein to LRP5 or LRP6. As defined herein“modulate” means enhancing or preventing the binding and/or stability ofbinding of a protein to LRP5 or LRP6, preferably a YWTD repeat domain ofLRP5 or LRP6. The protein may include but is not limited to a member ofthe Dkk family, a member of the Wnt family, sclerostin, or a PA receptor(e.g., TEM8 or CMG2).

For instance, NCI 8642 (also referred to as IIIC3), has the structure:

Retaining the core structure, and indicating where various substitutionscan take place, a generalized formula for a family of analogues of thiscompound can be as follows (I):

wherein at least one of R¹, R³, R⁴, R⁶, R⁸, R¹¹, R¹² or R¹³ is ahydrogen atom and wherein at least one of R¹, R³, R⁴, R⁶, R⁸, R¹¹, R¹²or R¹³ comprises an atom other than a hydrogen atom. In a particularembodiment, R¹, R³, R⁴, R⁶, R⁸, R¹¹, R¹², and R¹³ independently comprisehydrogen, oxygen, hydroxy, a halogen, a linear or branched (C₁-C₁₆)alkyl group, a substituted linear or branched (C₁-C₁₆) alkyl group, acycloalkyl group, a substituted cycloalkyl group, a heterocyclic group,a substituted heterocyclic group, an aryl alkyl group, a substitutedaryl alkyl group, a heteroarylalkyl group, a substitutedheteroararylalkyl group, an alkoxy group, a substituted alkoxy group, analkene group, a substituted alkene group, an acyl group, an amine group,an amide group, a nitrate, a nitrate ester, a carboxyl group, a carboxylester, a sulfide, a sulfoxide, a sulfonate, a sulfonate ester, asulfone, a sulfonamide, a phosphate, a phosphate ester, a phosphonate, aphosphonate ester, a phosphamide, a phosphoramide, a thiophosphate, athiophosphate ester, a thiophosphonate, or a thiophosphonate ester,wherein R¹ and R¹¹, R¹¹ and R¹², R¹² and R³, R³ and R⁴, R¹³ and R⁶ mayindependently be fused together to form one or more rings, or anycombination of the foregoing. When the nitrogen of the amine groupcomprising R¹¹ and R¹² is charged and further comprises R¹⁵, wherein R¹⁵is as described previously for R¹, R³, R⁴, R⁶, R⁸, R¹¹, R¹² and R¹³. Ina particular embodiment, the compound has the structure (VIII):

wherein R¹³ is a linear or branched alkyl group or substituted orunsubstituted cycloalkyl group. In a most particular embodiment R¹³ is alinear or branched C₂₋₄ group. In another particular embodiment R13 is acycloalkyl C3-8 group.

This core compound can be generalized further by retaining the ringstructure and allowing substitutions for the carboxyl or ester groupshown in the structure above, giving a formula (II) for a series ofother analogues as follows:

wherein at least one of R¹, R³, R⁴, R⁶, R⁸, R¹¹, R¹², R¹³ or R¹⁴ is ahydrogen atom and wherein at least one of R¹, R³, R⁴, R⁶, R⁸, R¹¹, R¹²,R¹³ or R¹⁴ comprises an atom other than a hydrogen atom

In a particular embodiment, R¹, R³, R⁴, R⁶, R⁸, R¹¹, R¹², R¹³ and R¹⁴independently comprise hydrogen, oxygen, hydroxy, a halogen, a linear orbranched (C₁-C₁₆) alkyl group, a substituted linear or branched (C₁-C₁₆)alkyl group, a cycloalkyl group, a substituted cycloalkyl group, aheterocyclic group, a substituted heterocyclic group, an aralalkylgroup, a substituted arylalkyl group, a heteroarylalkyl group, asubstituted heteroarylalkyl group, an alkoxy group, a substituted alkoxygroup, an alkene group, a substituted alkene group, an acyl group, anamine group, an amide group, a nitrate, a nitrate ester, a carboxylgroup, a carboxyl ester, a sulfide, a sulfoxide, a sulfonate, asulfonate ester, a sulfone, a sulfonamide, a phosphate, a phosphateester, a phosphonate, a phosphonate ester, a phosphamide, aphosphoramide, a thiophosphate, a R¹¹ and R¹², R¹² and R³, R³ and R⁴,R¹³ and R⁶ may independently be fused together to form one or morethiophosphate ester, a thiophosphonate, or a thiophosphonate ester,wherein R¹ and R¹¹, rings, or any combination of the foregoing.

In a particular embodiment the compound has the structure (VII):

wherein R¹³ and R¹⁴ are each independently H or a linear or branchedalkyl group.

In a more particular embodiment, R¹³ and R¹⁴ are independently H or alinear or branched C₁₋₅ linear or branched alkyl group. In most specificembodiments, R¹³ is H and R¹⁴ is CH₃ groups. (Enz M14); R¹³ and R¹⁴ areCH₃ groups (Enz M15); R¹³ are CH₃ groups and wherein R¹⁴ is C(CH₃)₃ (EnzM25); R¹³ is H and R¹⁴ is (CH₂)₂CH(CH₃)₂. (Enz M35); R¹³ is H, whereinR¹¹ and R¹² are CH₃ groups and wherein R¹⁴ is CH₂CH(CH₃)(CH₂CH₃). (EnzM39).

The compound encompassed by (VII) may be obtained by

(a) reacting gallocyanine with an agent to replace the COOH group ongallocyanine with a leaving group;

(b) reacting the compound obtained in step (a) with an alkyl amine toobtain said compound (VII).

The invention is further directed to a novel compound having thestructure (VI):

wherein R¹⁵ is a linear or branched alkyl group. In a particularembodiment, R¹⁵ is a linear or branched C₁₋₅ alkyl group. In mostspecific embodiments, R¹⁵ is a methyl group (Enz M01); R¹⁵ is an ethylgroup (EnzM02); R¹⁵ is a propyl group (EnzM03); R¹⁵ is CH₂C(CH₃)₃(EnzM12).

This compound may be obtained by reacting gallocyanine with an alkylhalide under conditions promoting formation of said compound.

In a similar fashion, a series of compounds that may be of interest maybe designed using IC15 and IC5 as starting points:

As described previously, the common anthra-9,10-quinone structure inthese two compounds was used in a secondary screening with UNITY™followed by docking with FlexX™ and biological assays. This led to theidentification of IIC8, IIC10, IIC18 and IIC19 (all sharing theanthra-9,10-quinone) as demonstrating effects upon Wnt activity. Thus,in this instance a family of analogues could have the generalizedstructure (III):

wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷ or R⁸ is a hydrogenatom and wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷ or R⁸comprises an atom other than a hydrogen atom. In a preferred embodiment,R¹, R², R³, R⁴, R⁶, R⁶, R⁷, R⁸ independently comprise hydrogen, oxygen,hydroxy, a halogen, a linear or branched (C₁-C₁₆) alkyl group, asubstituted linear or branched (C₁-C₁₆) alkyl group, a cycloalkyl group,a substituted cycloalkyl group, a heterocyclic group, a substitutedheterocyclic group, an aralalkyl group, a substituted aralalkyl group, aheteroarylalkyl group, a substituted heteroaryllalkyl group, an alkoxygroup, a substituted alkoxy group, an alkene group, a substituted alkenegroup, an acyl group, an amine group, an amide group, a nitrate, anitrate ester, a carboxyl group, a carboxyl ester, a sulfide, asulfoxide, a sulfonate, a sulfonate ester, a sulfone, a sulfonamide, aphosphate, a phosphate ester, a phosphonate, a phosphonate ester, aphosphamide, a phosphoramide, a thiophosphate, a thiophosphate ester, athiophosphonate, or a thiophosphonate ester, wherein R¹ and R², R² andR³, R³ and R⁴, R⁵ and R⁶, R⁶ and R⁷, R⁷ and R⁸ may independently befused together to form one or more rings, or any combination of theforegoing.

The invention further provides a family of compounds that could be basedupon the secondary screening described above where IIC15 (structureshown below) showed interesting effects upon Wnt regulation.

Other compounds that share common structural elements with this compoundand which demonstrate effects upon Wnt activity are IIC1, IIC2, IIC7 andIIIC10, the structures of which are shown in Table IV.

Thus, the invention provides for compounds having the structure (IV):

wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ is ahydrogen atom and wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷ orR⁸ comprises an atom other than a hydrogen atom. In a specificembodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ or R¹⁰ independentlycomprise hydrogen, oxygen, hydroxy, a halogen, a linear or branched(C₁-C₁₆) alkyl group, a substituted linear or branched (C₁-C₁₆) alkylgroup, a cycloalkyl group, a substituted cycloalkyl group, aheterocyclic group, a substituted heterocyclic group, an arylalkylgroup, a substituted arylalkyl group, a heteroarylalkyl group, asubstituted heteroarylalkyl group, an alkoxy group, a substituted alkoxygroup, an alkene group, a substituted alkene group, an acyl group, anamine group, an amide group, a nitrate, a nitrate ester, a carboxylgroup, a carboxyl ester, a sulfide, a sulfoxide, a sulfonate, asulfonate ester, a sulfone, a sulfonamide, a phosphate, a phosphateester, a phosphonate, a phosphonate ester, a phosphamide, aphosphoramide, a thiophosphate, a thiophosphate ester, athiophosphonate, or a thiophosphonate ester, wherein R¹ and R², R² andR³, R⁴ and R⁵, R³ and R⁴, R⁶ and R⁷, R⁷ and R⁸, R⁸ and R⁹ and R⁹ and R¹⁰may independently be fused together to form one or more rings, or anycombination of the foregoing.

It should be noted that the R groups on the aromatic rings of these corestructures may be fused together to form more complex ring structures.Thus, for instance, carbon chains of R² and R³ could be joined togetherto give a series of compounds having the structure (V):

wherein at least one of R¹, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ is a hydrogenatom and wherein at least one of R¹, R⁴, R⁵, R⁶, R⁷ or R⁸, R⁹, R¹⁰, R¹¹,R¹², R¹³ comprises an atom other than a hydrogen atom. In a specificembodiment, R¹, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴independently comprise hydrogen, oxygen, hydroxy, a halogen, a linear orbranched (C₁-C₁₆) alkyl group, a substituted linear or branched (C₁-C₁₆)alkyl group, a cycloalkyl group, a substituted cycloalkyl group, aheterocyclic group, a substituted heterocyclic group, an arylalkylgroup, a substituted arylalkyl group, a heteroarylalkyl group, asubstituted heteroarylalkyl group, an alkoxy group, a substituted alkoxygroup, an alkene group, a substituted alkene group, an acyl group, anamine group, an amide group, a nitrate, a nitrate ester, a carboxylgroup, a carboxyl ester, a sulfide, a sulfoxide, a sulfonate, asulfonate ester, a sulfone, a sulfonamide, a phosphate, a phosphateester, a phosphonate, a phosphonate ester, a phosphamide, aphosphoramide, a thiophosphate, a thiophosphate ester, athiophosphonate, or a thiophosphonate ester, wherein R¹ and R¹¹, R¹¹ andR¹², R¹² and R¹³, R¹³ and R¹⁴, R¹⁴ and R⁴, R⁴ and R⁵, R⁶ and R⁷, R⁷ andR⁸, R⁸ and R⁹ and R⁹ and R¹⁰ may independently be fused together to formone or more rings, or any combination of the foregoing.

Examples of tested compounds that share this structure are IIC1 and IIC2mentioned above.

The core compounds disclosed hereinabove can be combined to formcompounds that are an amalgam that span various interaction points ofthe various core compounds described above. For instance, IC13, whichwas shown to affect Wnt activity, has two of the III moieties linkedtogether and in combination with the linkage between them it alsocomprises an analogue structure derived from the IV moiety. Thestructure of this hybrid molecule is shown below:

Other Protein Surface Targets of the Wnt Signaling Pathway

There are numerous proteins involved in the Wnt signaling pathway thatparticipate in protein/protein interactions which can be affected by theadministration of pharmacological compounds. Examples of variousproteins that participate in protein/protein interactions in the LRP516and/or canonical and non-canonical Wnt pathways are described innumerous references (82, 68, 83, 81, 84, 85, 86, and 87). For a giventarget protein of this group, there can be a variety of differentproteins that interact with the target protein. These binding partnersmay bind to different sites on the target protein or two differentbinding partners may share the same site or overlapping sites on thetarget protein. The methods of the present invention can be applied toeither participant in each interaction pair and appropriate structuresand domains can be identified by any means known to those skilled in theart. These means can include but not be limited to, construction ofclones comprising the various portions of target proteins (extracellularand intracellular domains, for example) and testing for binding withvarious deletion mutants of the interacting protein partner; randommutation of the target protein and measuring effects upon Wnt activityor some other biological marker followed by sequencing of interestingmutants; alanine scanning to identify critical amino acids involved inthe function of protein/protein interactions of the selected interactingpair, construction of various deletion mutants of the target protein andmeasuring their ability to influence Wnt activity or some otherbiological marker; construction of AP fusion versions of deletionmutants of the target protein; the use of analogous proteins to preparemodels of protein structures; the use of peptide libraries to determinepeptide sequences that bind to particular targets; X-ray crystallographyand NMR analysis. As described above, many of these structure and sitesare known in the art and the particular site on a target protein may bechosen for virtual screening and assayed by various means. For otherprotein/protein interactions, the existence of the interaction is knownbut the particular site needs further investigation by any of the meansdescribed above. Examples of protein targets and their binding site foran interacting protein are given below and include but are not limitedto:

Structural Target Binding sites for: Wnt LRP5/6, Dkk, Frizzled, sFRPs,Wif-1, Cerebrus LRP5/6 Wnt, Axin, Boca, mesd, Frat1, Wise, SOST,TEM8/ATR, CMG2 Dkk Wnt, Kremen Dishevelled Frizzled, Axin, nakedFrizzled Wnt, Disheveled, Norrin Beta-catenin APC, LEF/TCF, E-cadherin,Axin LEF/TCF Beta-catenin

In protein/protein interactions, there is a site on each constituentthat participates in the interaction, and thus either surface can serveas a potential target for virtual screening. There are at least twobenefits achieved by this approach. In the first place, the potentialpharmacopia of compounds influencing Dkk/LRP reactions is now widened bythe inclusion of entirely new families of compounds identified by anaffinity for Dkk. Secondly, the binding domains of proteins that areinvolved in signal generation tend to be multivalent. For instance, thebinding domains on LRP5/6 are used by a number of different proteinsthat vary in their purpose and effect. With regard to the presentexample, Dkk may bind to the second and third domains of LRP5/6 (79),but it has also recently been shown that sclerostin can bind to thefirst and second LRP5/6 domains (80) and that one of these domainsserves as a binding site for the toxic effects of anthrax (81).

Thus, even when a particular drug is targeted to a single site on theLRP5/6 receptor, there may be a number of different signal generatingproteins other than Dkk that may be affected by a drug binding to thatsite. These effects may also be beneficial or they may be neutral oreven deleterious thus affecting the use of a compound for therapeuticuse. By the same token, when the corresponding LRP5/6 binding site onDkk is chosen as a target, there may be a variety of otherprotein/protein interactions that also use this locus. As such, when adrug is identified that binds to this site, the particular proteins thatmay be influenced by a drug selected for binding to the LRP/Dkk bindingsite on Dkk may represent a different group of proteins from thoseaffected by a drug selected for binding to the LRP/Dkk binding site onLRP5/6.

In addition to targeting the other side of the Wnt/Dkk reaction, otherprotein/protein interactions involved in Wnt signaling have also beendescribed as potential candidates for the foregoing processes. In asimilar fashion, they achieve some of the benefits described above. Forinstance, similar to choosing Dkk instead of LRP5/6 as a target, the useof a different protein target should allow the discovery of an entirelydifferent set of compounds that can affect the Wnt signaling pathway. Inthis case, it is possible that the same effect may be achieved(influencing Wnt signaling) but a different part of the pathway isaffected. It also should be understood that a dual discovery program canbe carried out where each partner of a protein/protein interaction is acandidate for drug discovery. Secondly, as described earlier, a compoundthat influences one particular protein/protein interaction is alsolikely to affect other protein/protein interactions that were notnecessarily the primary target. Thus it may prove that some members ofthe Wnt signaling pathways may be more specific than others in achievingdesirable effects. In addition to the particular protein/proteininteractions that were specified earlier, it should be understood thatall proteins that participate in protein/protein interactions for Wntsignaling can potentially be candidates for carrying out procedures ofthe present invention. These protein targets can participate in bothcanonical and non-canonical signaling pathways or they may be restrictedto one pathway or the other.

As noted above, the compounds used in the compositions and methods ofthe present invention may be used to modulate pathophysiologicalprocesses. As defined herein “modulate” includes but is not limited toaltering the amount of or rate of a particular process, adjusting aprocess, or adjusting to or keeping in proper measure or proportion.

The present invention can be applied to numerous systems that areaffected by or dependent upon the canonical or non-canonical Wntsignaling pathway. These may be diseases or conditions that are causedby alterations or defects in the Wnt signaling pathway where adjustmentsin Wnt activity may alleviate these diseases or conditions.Alternatively, there may be disease or conditions that are not caused byproblems with Wnt activity per se but curative or therapeutic effectsmay be achieved by manipulations of Wnt activity. As describedpreviously in U.S. Patent Application Serial No. 20050196349, U.S.Patent Application Serial No. 20050261181 and U.S. Patent ApplicationSerial No. 20060030523, all of which are incorporated herein byreference, examples of applications where manipulations of the Wntsignaling system may provide beneficial effects can include but not belimited to influencing bone formation and remodeling as well as treatingtumors and abnormal growths.

As described above, the compounds that have been identified through thepresent invention may also find application in curative or preventivemeans in tumors and abnormal growth. Although it would be understoodfrom the previous passage that tumors or abnormal growth of bone cellsor bone tissues could be a target for these compounds, Wnt activity ismore widespread and a factor in abnormal growth of other cells andtissues as well. It should be pointed out that when the first Wnt genewas isolated, it was considered to be a proto-oncogene (92) and named“int-1” due to its propensity to cause tumors when mouse mammary virusintegrated into this site. It was only years later, when the role ofthis family of genes in embryonic development became clear, that it wasrenamed “Wnt” (39). Since genes involved in the Wnt pathway areexpressed in a wide variety of tissues, there is an equal connectionwith a large variety of different tumor types associated with changes inWnt pathway expression. Additionally, there are different effectsdepending upon the particular type of cancer. For instance, evidence hasbeen shown that Wnt 5A could be a tumor suppressor in hematopoieticcells (93) and on the other hand, Wnt 5A may promote motility andinvasiveness in melanomas thereby potentially having a role inmetastasis (94). For reviews on the interactions of the Wnt pathway andtumorigenesis or tumor maintenance as well as the numerous types ofcancers where these effects are seen (see cited references 95, 96, 97,66, 98, and 99). It should also be understood that a linkage of effectsby Wnt on cancer and control of bone growth is not a phenomenon that isrestricted to bone cancers. Multiple myeloma, which is essentially ahematologic cancer, also has indirect effects upon bone growth whereincreased fragility is one of the hallmarks of the disease. This lattereffect has been traced to increased levels of Wnt inhibitors Dkk (100)and sFRP-2 (101) in myeloma cells.

Disease conditions associated with alterations in the Wnt pathwaysignaling system are not restricted to just tumors or abnormal bonegrowth. Genetic defects in genes involved in expression of components ofthe Wnt signaling system exhibit defects in various organs and systems.Even for the same gene, the particular site or even the particularmutation can affect the particular phenotypic expression of the defect.As described earlier, certain mutations in LRP5 lead to increased bonegrowth and other mutations lead to diminished bone growth. Some geneticdiseases are likely related to developmental processes that are affectedby a defect in a constituent of the Wnt signaling system. For instance,a mutation in either LRP5 or FZD4 can lead to Familial ExudativeVitreoretinopathy characterized by a lack of vascularization of theretina (102). In a more radical instance, there is a complete failure togenerate limbs in a condition called Tetra-amelia which is caused by adefect in Wnt3 (103). Other genetic mutations may affect the role of Wntsignaling in homeostatic processes that are post-embryonic in nature.For instance, the defects that led to either increased or decreased bonemass as well as tumorigenic events are ongoing post-birth processes.However, as described above, these are not the only homeostaticprocesses affected by Wnt signaling. The possibility of a mutation in aWnt pathway gene affecting diabetes had been previously indicated byresults from mice deficient in both copies of LRP5. Although these micewere cited earlier in the context of a potential model for osteoporosis,further studies showed that this strain exhibited other phenotypiccharacteristics as well including increased serum cholesterol levels anda markedly impaired glucose tolerance response (20). The latter resultis not surprising since the LRP5 gene was initially mapped and cloneddue to its proximity to the IDDM4 region which has been associated withdiabetes (18). In addition, a recent study (104) has shown an increasedlikelihood of development of Type 2 Diabetes in individuals with anallele in the TCF7L2 gene (formerly referred to as TCF4) with riskfactors of 1.45 and 2.41 for heterozygous and homozygous conditions,respectively. Other diseases or conditions that have been found to beassociated with various members of the Wnt signaling system includedevelopment of polycystic kidney disease (105), renal fibrosis (106),pulmonary fibrosis (107), aggressive fibramotosis (108), andschizophrenia (109). (For reviews on the relationship between Wntsignaling pathways and disease, see cited references 63 and 66).

As such, any disease processes that are either caused by changes in Wntsignaling or show differences in Wnt signaling as a result of thepresence of a particular disease or condition may find use with thecompounds that are identified through the use of the present invention.

The compounds used in the method of the present invention modulate, andin particular, attenuate the Mesd-LRP5 interaction, resulting in lessLRP5 receptors present at the cell surface which may lead to an increasein bone density through bone formation or bone remodeling.

Dkk acts as a Wnt antagonist when it binds to, or interacts with, thethird domain of the LRP5 receptor. Compounds have been identified thatinhibit the Dkk-LRP5 interaction to promote bone formation orremodeling. As described in the Examples below, one compound, NCI366218(IIC8) has been found to stimulate osteoblast differentiation in tissueculture models. Wnt and Dkk have been shown to regulate the growth anddifferentiation of mesenchymal stem cells. Compounds have beenidentified which function as mesenchymyl stem cell regulators for theregulation of bone formation and for the development and differentiationof hemaetopoietic stem cells.

Wnt has been shown to regulate the growth and differentiation ofhematopoietic stem cells. Compounds have been identified which functionas hemaetopoietic stem cell regulators for the regulation of boneformation and for the proliferation and expansion of stem cells in vivoand in vitro.

Compositions

The compound(s) used in the method of the present invention may beformulated into a composition, most notably a pharmaceuticalcomposition. Such a composition typically contains from about 0.1 to 90%by weight of a metabolic intermediate of the invention formulated inand/or with a pharmaceutically acceptable carrier or excipient.Pharmaceutical formulation is a well-established art, and is furtherdescribed in Gennaro (ed.), Remington: The Science and Practice ofPharmacy, 20th ed., Lippincott, Williams & Wilkins (2000); Ansel et al.,Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed.,Lippincott Williams & Wilkins (1999); and Kibbe (ed.), Handbook ofPharmaceutical Excipients American Pharmaceutical Association, 3rd ed.(2000).

Briefly, formulation of the pharmaceutical compositions of the presentinvention will depend upon the route chosen for administration. Thepharmaceutical compositions utilized in this invention can beadministered by various routes including both enteral and parenteralroutes, including oral, intravenous, intramuscular, subcutaneous,inhalation, intrathecal, intraventricular, transmucosal, transdermal,intranasal, intraperitoneal, and intrapulmonary. The pharmaceuticalcomposition may comprise one or more agents of the present invention.

Oral dosage forms can be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient. Solid formulations of the compositions fororal administration can contain suitable carriers or excipients, such ascarbohydrate or protein fillers, such as sugars, including lactose,sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato,or other plants; cellulose, such as methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, ormicrocrystalline cellulose; gums including arabic and tragacanth;proteins such as gelatin and collagen; inorganics, such as kaolin,calcium carbonate, dicalcium phosphate, sodium chloride; and otheragents such as acacia and alginic acid. Agents that facilitatedisintegration and/or solubilization can be added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate, microcrystalline cellulose, cornstarch, sodium starch glycolate, and alginic acid. Tablet binders thatcan be used include acacia, methylcellulose, carboxymethylcellulose,polyvinylpyrrolidone (Povidon™), hydroxypropyl methylcellulose, sucrose,starch and ethylcellulose. Lubricants that can be used include magnesiumstearates, stearic acid, silicone fluid, talc, waxes, oils, andcolloidal silica. Fillers, agents that facilitate disintegration and/orsolubilization, tablet binders and lubricants, including theaforementioned, can be used singly or in combination. Solid oral dosageforms need not be uniform throughout. For example, dragee cores can beused in conjunction with suitable coatings, such as concentrated sugarsolutions, which can also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures.

Oral dosage forms of the present invention include push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and acoating, such as glycerol or sorbitol. Push-fit capsules can containactive ingredients mixed with a filler or binders, such as lactose orstarches, lubricants, such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds can bedissolved or suspended in suitable liquids, such as fatty oils, liquid,or liquid polyethylene glycol with or without stabilizers. Additionally,dyestuffs or pigments can be added to the tablets or dragee coatings forproduct identification or to characterize the quantity of activecompound, i.e., dosage.

Liquid formulations of the pharmaceutical compositions for oral(enteral) administration are prepared in water or other aqueous vehiclesand can contain various suspending agents such as methylcellulose,alginates, tragacanth, pectin, kelgin, carrageenan, acacia,polyvinylpyrrolidone, and polyvinyl alcohol. The liquid formulations canalso include solutions, emulsions, syrups and elixirs containing,together with the active compound(s), wetting agents, sweeteners, andcoloring and flavoring agents.

The pharmaceutical compositions of the present invention can also beformulated for parenteral administration. Formulations for parenteraladministration can be in the form of aqueous or non-aqueous isotonicsterile injection solutions or suspensions.

For intravenous injection, water soluble versions of the compounds ofthe present invention are formulated in, or if provided as a lyophilate,mixed with, a physiologically acceptable fluid vehicle, such as 5%dextrose (“D5”), physiologically buffered saline, 0.9% saline, Hanks'solution, or Ringer's solution. Intravenous formulations may includecarriers, excipients or stabilizers including, without limitation,calcium, human serum albumin, citrate, acetate, calcium chloride,carbonate, and other salts.

Intramuscular preparations, e.g. a sterile formulation of a suitablesoluble salt form of the compounds of the present invention, can bedissolved and administered in a pharmaceutical excipient such asWater-for-Injection, 0.9% saline, or 5% glucose solution. Alternatively,a suitable insoluble form of the compound can be prepared andadministered as a suspension in an aqueous base or a pharmaceuticallyacceptable oil base, such as an ester of a long chain fatty acid (e.g.,ethyl oleate), fatty oils such as sesame oil, triglycerides, orliposomes.

Parenteral formulations of the compositions can contain various carrierssuch as vegetable oils, dimethylacetamide, dimethylformamide, ethyllactate, ethyl carbonate, isopropyl myristate, ethanol, polyols(glycerol, propylene glycol, liquid polyethylene glycol, and the like).

Aqueous injection suspensions can also contain substances that increasethe viscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Non-lipid polycationic amino polymers can also beused for delivery. Optionally, the suspension can also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Pharmaceutical compositions of the present invention can also beformulated to permit injectable, long-term, deposition. Injectable depotforms may be made by forming microencapsulated matrices of the compoundin biodegradable polymers such as polylactide-polyglycolide. Dependingupon the ratio of drug to polymer and the nature of the particularpolymer employed, the rate of drug release can be controlled. Examplesof other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in microemulsions that are compatible with bodytissues.

Inhalation formulations can also readily be formulated. For inhalation,various powder and liquid formulations can be prepared. For aerosolpreparations, a sterile formulation of the compound or salt form of thecompound may be used in inhalers, such as metered dose inhalers, andnebulizers. Aerosolized forms may be especially useful for treatingrespiratory disorders.

Alternatively, the compounds of the present invention can be in powderform for reconstitution in the appropriate pharmaceutically acceptablecarrier at the time of delivery.

The pharmaceutically active compound in the pharmaceutical compositionsof the present invention can be provided as the salt of a variety ofacids, including but not limited to hydrochloric, sulfuric, acetic,lactic, tartaric, malic, and succinic acid. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms.

After pharmaceutical compositions have been prepared, they are packagedin an appropriate container and labeled for treatment of an indicatedcondition.

The active compound will be present in an amount effective to achievethe intended purpose. The determination of an effective dose is wellwithin the capability of those skilled in the art.

The therapeutically effective dose of the compound(s) used in thepresent invention can be estimated initially by in vitro tests, such ascell culture assays, followed by assay in model animals, usually mice,rats, rabbits, dogs, or pigs. The animal model can also be used todetermine an initial preferred concentration range and route ofadministration.

For example, the ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population) can bedetermined in one or more cell culture of animal model systems. The doseratio of toxic to therapeutic effects is the therapeutic index, whichcan be expressed as LD₅₀/ED₅₀. Pharmaceutical compositions that exhibitlarge therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies are usedin formulating an initial dosage range for human use, and preferablyprovide a range of circulating concentrations that includes the ED₅₀with little or no toxicity. After administration, or between successiveadministrations, the circulating concentration of active agent varieswithin this range depending upon pharmacokinetic factors well known inthe art, such as the dosage form employed, sensitivity of the patient,and the route of administration.

The exact dosage will be determined by the practitioner, in light offactors specific to the subject requiring treatment. Factors that can betaken into account by the practitioner include the severity of thedisease state, general health of the subject, age, weight, gender of thesubject, diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long-acting pharmaceutical compositions can be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration. Ina particular embodiment, the daily dosage is about 0.01 mg to 30 mg/kgof body weight of the patient (e.g., 1 mg/kg to 5 mg/kg). Thepharmaceutical formulation can be administered in multiple doses perday, if desired, to achieve the total desired daily dose.

Conventional methods, known to those of ordinary skill in the art ofmedicine, can be used to administer the pharmaceutical formulation(s) ofthe present invention to the patient. The pharmaceutical compositions ofthe present invention can be administered alone, or in combination withother therapeutic agents or interventions. Specifically, thecompositions of the present invention may further comprise a pluralityof agents of the present invention. The treatment of a disease in any ofthe described methods results in a change in the number or function ofregulatory, immune-regulatory or NKT cells. This change encompasses areduction, inhibition, or decrease in the number or function of thecells. This inhibition may be caused by the competitive displacement ofactivating elements from the CD1d molecule. A change may also include astimulation or increase in the number or function of the cells. Thisstimulation may be caused by increased binding of the activatingelements from the CD1d molecule

Synergism and Indirect Effects

When tested in biological assays, the compounds selected by virtualscreening for binding to LRP5/6 showed various effects upon: a) Lefactivity; b) enhancement of lef activity by Wnt; and c) repression ofWnt enhancement by Dkk (as seen in Table I). Thus, the compositions ofthe present invention may comprise two or more compounds that modulatepathophysiological processes in a subject.

It is well known that the binding of a small molecule to a protein canalter the conformation of the protein. For instance, numerouscrystallographic studies have been pursued to study the conformation ofproteins with and without substrates to elicit details on binding sitesand catalytic mechanisms of enzymes. As such, it could easily beunderstood that binding of a compound can alter the conformation of aprotein such that it can enhance its activity or reduce its activity butalso it can be understood that even a change in the conformation mayhave no effect upon the activity per se. However, it can be seen that aprotein with a small molecule bound to it is not the same proteinwithout the molecule. Thus small molecules that are able to bind to aparticular target protein but had no effect upon the native protein mayhave effects on the target protein when it has been forced into adifferent conformation. The conjunction of two different compounds boundto the same protein target may consist of one molecule that hadpreviously shown to change behavior of the system and a second thatshowed no effect when used alone or it is possible that it consists oftwo proteins, neither of which shows activity alone. The same assaysthat are used to test the individual compounds may then be used in thepreviously described assays with both compounds present, i.e. change inLef activity, response of Lef activity to the presence of Wnt (Wntactivation), response to inhibition of Wnt by Dkk and lastly changes inthe affinity of LRP5 and Dkk-AP. A series of reactions may be carriedout where a compound such as gallocyanine or enzoM01 is used in thepresence of other compounds that have been selected for binding toLRP5/6. On the other hand, a wider search may be employed where a matrixof various combinations of compounds is carried out.

A further aspect of the present invention is to take advantage ofcomplementary or synergistic effects of administration of a mixture oftwo or more pharmacological agents. Another possibility is for two ormore drugs that in combination at lower dosages can provide the sametherapeutic effects as one of the drugs at a normal dosage. Theadvantage for this method may be reducing the expenses of the drug orthere may be undesirable side effects that may be reduced by the use ofa lower dosage.

EXAMPLES Materials and Methods

Cell Culture, Transfection, Preparation of CM, and Luciferase Assay

Human embryonic kidney cell (HEK) line A293T and mouse fibroblast cellline NIH3T3 were maintained and transfected as previously described (1).Pre-osteoblast cell lines 2T3 and MC3T3 were cultured in a-MEMcontaining 10% FCS. For luciferase assays, cells in 24-well plates wereseeded at 5×10⁴ cells/well and transfected with 0.5 μg DNA/well usingLipofectamine Plus (Invitrogen, CA), as suggested by the manufacturer.The LacZ plasmid was usually used to make DNA concentrations equal foreach transfection. Cell extracts were collected 24 hours aftertransfection. Luciferase assays were performed as previously described(1, 2). Luminescence intensity was normalized against fluorescenceintensity of GFP. For the preparation of Dkk1-AP containing CM, HEKcells were seeded in 6 well-plates at 4×10⁵ cells/well and transfectedwith 1 μg DNA/well. CMs were collected 48 hours after transfection.

Construction of Expression Plasmids and Mutagenesis

The wild-type and mutant forms of human LRP5, LRP6, mouse Wnt1, Dkk1,and Dkk2 were generated by PCR using the high fidelity thermostable DNApolymerase Pfu Ultra (Stratagene, CA). HA or Flag epitope tags wereintroduced to the C-termini of the full-length and mutant molecules. Theexpression of these molecules was driven by a CMV promoter. The LEF-1reporter gene constructs were obtained from an outside source (3).

Dkk1-AP Binding Assay and Immunoprecipitation Assay

HEK cells in 24-well plates were transfected with LRP5 and its mutants.One day later, cells were washed with cold washing buffer (HBBScontaining BSA and NaN₃) and incubated on ice with mouse Dkk1-APconditioned medium for two hours. The cells were then washed three timeswith washing buffer and lysed. The lysates were heated at 65° C. for 10minutes, and their AP activity was determined using a Tropixluminescence AP assay kit. The immunoprecipitation assays were carriedout as previously described (4).

Biotinylation of Cell Surface Proteins

HEK cells were transfected with LacZ, LRP5, and LRP5_(G171V) expressionplasmids. The cells were labeled with 0.5 mg/ml sulfo-NHS-biotin(Pierce) in ice-cold PBS, washed and lysed as previously described (5).The cell lysate was immunoprecipitated with an anti-HA antibody andA/G-agarose protein.

Primary Osteoblast Cultures

Bone marrow stromal (BMS) osteoblast cultures from 3 month old mice weregenerated as previously described (6). The cells were induced to undergoosteogenic differentiation in the presence of 10 nM Dexamethasone, 8 mMβ-Glycerophosphate, and 50 ug/ml ascorbic acid. The media was changedevery two days.

Homology Modeling

A homology model of the third YWTD-EGF domain of LRP5 was built with ICM(Molsoft L.L.C., La Jolla, Calif.) using sequences obtained from theSwiss-Prot/TrEMBL database (Entry Name Q9UP66 (8)). The LDL receptor(Low-Density Lipoprotein) YWTD-EGF domain (PDB code 1IJQ (9)) was chosenas the template.

Virtual Screening

The UNITY™ program (Tripos, Inc.) was used to screen the National CancerInstitute (NCI) database for chemical compounds that were able to fitinto the cavity formed by six-propellers at the end with Glu456. Thecandidate compounds were then docked into the Dkk1 binding cavities ofthe LRP5 domains using the FlexX™ program (Tripos, Inc.) for energyminimization (10). The chemical compounds displaying the highest bindingaffinities in the calculations were obtained from the Drug Synthesis &Chemistry Branch, Developmental Therapeutics Program, Division of CancerTreatment and Diagnosis, National Cancer Institute, for furtherexperimental tests. Second and third rounds of screenings were carriedout based on the results of biochemical assays.

Evaluation of the Effects of Compounds on Bone

IIIC3 was dissolved in DMSO and diluted 1:1000 into PBS at aconcentration of 0.44 mg/ml. For calvarial local injection, fifteenmicro-liters of IIIC3 (0.22 mg/Kg/day), control vehicle, or positivecontrol (b-FGF, 12.5 ug/Kg/day) were injected into the subcutaneoustissue over the right side of the calvaria of four weeks old CD-1 micethree times a day for 5 days using a injection method describedpreviously (74, 75). Calvarias were collected 22 days after the firstinjection and fixed for sectioning. For systemic administration, IIIC3(3 mg/Kg/d) and control vehicle were injected intraperitoneally into 2month old C57B1 mice (n=19) once a day for 7 days. Then mice were restedfor three weeks, and the treatment was repeated once. Three weeks afterthe last injection, the mice were anesthetized, and total femoral andwhole body bone mineral content (BMC; grams) and bone area (squarecentimeters) were measured using the PIXImus small animal DXA system(GE-Lunar, Madison, Wis.), and BMD was calculated. Heads were excludedfor the whole body measurement. These mice were also weighted at thetime.

Wnt Activity Reporter Gene Assay

The assay was carried out as previously described (30, 78). In brief,cells in 24-well plates were seeded at 5×10⁴ cells/well and transfectedwith 0.1 mg GFP, 0.025 ug LEF-1, 0.075 ug LEF luciferase reporter gene,and 0.3 ug LacZ per well using Lipofectamine Plus (Invitrogen, CA), assuggested by the manufacturer. Compounds and conditioned mediums wereadded 24 hr after transfection. Six hours later, cell extracts werecollected, and luciferase assays were performed. Luminescence intensitywas normalized against fluorescence intensity of GFP.

Example 1 Deletion Mutants of LRP5

A set of PCR primers were designed, PCR reactions were carried out, andPCR fragments were sucloned into vectors to generate several LRP5deletion mutants. Deletion of the third and fourth domains (residues 646to 1198) resulted in LRP5R12; deletion of the first and second domains(residues 1 to 646) resulted in LRP5R34; and deletion of the thirddomain (residues 947 to 1198) resulted in LRP5R124. (see FIG. 1A).

Example 2 Domain I of LRP5 is Essential for Mesd-Mediated LRP5 FunctionExample 2.1 The G171V Mutation in the First Domain of LRP5 Disrupts LRP5Trafficking

Interaction of LRP5 with Mesd

HEK cells were transfected with expression plasmids, as indicated inFIG. 2A. One day later, the cells were lysed and immunoprecipitation wascarried out using an anti-Flag antibody. Mesd was Flag-tagged and allLRP5 molecules were HA-tagged. The results showed that the G171Vmutation of domain I disrupted the interactions of both LRP5 with Mesd(FIG. 2A, lanes 1 and 3) and R12 with Mesd (FIG. 2B, lanes 1 and 2),whereas the E721 mutation of domain III showed no effect on theinteraction (FIG. 2A, lanes 2 and 3).

LRP5 Mutants Do Not Efficiently Present Themselves to the Cell Surface

HEK cells were transfected with Mesd plasmids and expression plasmids,as indicated in FIG. 2B and FIG. 2C. R12TGV, R12T, R1-4 and R1-4GV (GV)are AP fusion proteins, which are LRP5 mutants lacking transmembranedomains that are secreted in the cell culture medium. One day later, theconditioned medium (CM) was collected and centrifuged at a high speed.The supernatant was either immunoprecipitated by an anti-HA antibody(FIG. 2C) or used for an AP assay (FIG. 2D). Cells were also lysed inthe SDS-PAGE sample buffer and analyzed by Western blotting (lowerpanels of FIGS. 2C&D). The results indicate that the G171V mutationattenuates the presentation of LRP5 to the cell surface.

Evaluation of Cell Surface LRP5 Levels

HEK cells were transfected with LacZ, wildtype HA-LRP5 or HA-LRP5G171Vexpression plasmids. The levels of cell surface LRP5 molecules weredetected by Western analysis using streptavidin-horse radish peroxidase(SA-HRP) after the cell surfaces were biotinylated and the LRP5molecules were precipitated with anti-HA antibody (FIG. 2E upper panel).The levels of LRP5 in the immunocomplexes are shown in the lower panel.These results show a decrease in cell surface presentation of the G171Vmutant.

Example 2.2 LRP5_(G171V) is Less Susceptible to Dkk1-Mediated Inhibitionof the Activity of Coexpressed Wnt

Effects of the G171V Mutation on Canonical Wnt Signaling Activity

HEK cells were transfected with plasmids, as indicated in FIG. 3A,together with LEF-1 expression plasmids, LEF-1 luciferase reporterplasmids and GFP expression plasmids. One day later, the cells werelysed. GFP levels and luciferase activity of the lysed cells weredetermined and normalized against GFP levels, as described in theMaterials & Methods. The activity from cells transfected with LacZ wastaken as 100% to establish the control. The expression of LRP5,LRP5_(G171V), LRP6, and LRP6_(G158V) was detected using an antibodyspecific to the HA tag carried by LRP5 proteins, or an anti-LRP6antibody (FIG. 3A). The results indicate that the HBM G171V mutation didnot lead to an increase in LEF-1-dependent transcriptional activitycompared to wildtype (Wt) LRP5 (LRP5_(Wt)) by itself or in transducingsignals for coexpressed Wnt. LEF-1 is a down-stream target transcriptionfactor of the canonical Wnt signaling pathway. Its activity, measured bya luciferase reporter gene assay, has been widely used to gauge thecanonical Wnt activity (12, 20). Thus, LRP5_(G171V) is neitherconstitutively active nor more competent in transducing Wnt signaling.Surprisingly, the corresponding mutation on LRP6, a substitution of aVal residue for Residue G-158, rendered it unable to act synergisticallywith Wnt-1 (FIG. 3A), likely inactivating the receptor.

Effects of the G171V Mutation on Canonical Signaling Activity StimulatedBy Coexpressed Wnt1

HEK cells were transfected with plasmids of LEF reporters, Wnt-1, Dkk1and Kremen in the presence of LRP5_(Wt) or LRP5_(G171V), as indicated inFIG. 3B. Human HEK cells were transfected with LacZ, or cotransfectedwith Dkk1, Kremen1 and Wnt1 in the presence of LRP5 or LRP5_(G171V). Inthe presence of both Kremen1 and DKK1, Wnt showed higher activity in HEKcells expressing LRP5_(G171V) than those expressing LRP5_(Wt) (FIG. 3B).These results indicate that the LRP5_(G171V) transduces more signalsthan the wild type in the presence of Dkk1. To ensure that thedifference was not a result of multi-plasmid transfection, the proteinexpression of Dkk1, Kremin1 and LRP5 (FIG. 3C) was examined. Similarresults of increased resistance to Dkk-mediated inhibition of autocrineWnt1 activity were also observed in NIH3T3 cells and two osteoblast-likecell lines, MC3T3 and 2T3.

Example 2.3 Binding of Dkk1-AP to LRP5 and LRP5 Mutants

HEK cells were transfected with Mesd plasmids and LRP5 plasmids, asindicated in FIG. 4, and incubated on ice with CM prepared from HEKcells expressing Dkk1-AP. The AP activity was determined in arbitraryunits (AU), as described in the Materials and Methods. The expression ofWt and mutant LRP5 molecules are shown in FIG. 4B. These resultsindicate that cells expressing the LRP5_(G171V) mutant show lessapparent Dkk binding than those expressing LRP5_(Wt) (FIG. 4A), which isconsistent with less LRP5_(G171V) on cell surfaces, shown in FIG. 2.

Example 3 Domain II of LRP5 is Required for Wnt Activity

HEK cells were transfected with the LEF activity reporter plasmids andexpression plasmids, as indicated in FIG. 5. Expression plasmidsLRP5R494Q and LRP5G479V are LRP5 receptors with point mutations in theirsecond domain. One day later, the cells were lysed. GFP levels andluciferase activity of the lysed cells were determined and normalizedagainst GFP levels, as described in the Materials & Methods. FIG. 5shows that LRP5R494Q and LRP5G479V can abolish Wnt signaling, ascompared to LRP5_(Wt). These results indicate that Domain II is requiredfor Wnt activity.

Example 4 Domain III is Required for Dkk-Mediated Inhibition Example 4.1Analysis of Domain III

The prevailing hypothesis for explaining why LRP5 G171V is lesssusceptible to Dkk1-mediated inhibition has been that the mutation coulddisrupt the interaction between LRP5 and Dkk1. It is reasonable tohypothesize that the first YWTD repeat domain that contains G171 isrequired for Dkk1-mediated antagonism. To test this hypothesis, two LRP5deletion mutants were generated: LRP5R12 with a deletion of the thirdand fourth YWTD repeat domains, and LRP5R34 with a deletion of the firstand second YWTD repeat domains (FIG. 1). To further delineate thesequence that is required for Dkk1-mediated inhibition, an additionalLRP5 mutant, LRP5R124, was generated in which the third YWTD repeatdomain was deleted (FIG. 1).

Functional Analysis of Domain III

HEK cells were transfected with the LEF activity reporter plasmids,Kremen1 plasmid and expression plasmids as indicated in the FIG. 6A. Theexpression of Wt LRP5 and its mutant molecules were shown in the FIG.6B. The result shows that LRP5R12 or LRP5R124, but not LRP5R34, couldstill potentiate Wnt-stimulated LEF-1 activity (FIG. 6A), suggestingthat LRP5R12 or LRP5R124 retains the Wnt coreceptor function. However,Dkk1 could not inhibit Wnt signaling when LRP5R12 or LRP5R124 waspresent (FIG. 6A). This suggests that the domain III is required forDkk1-mediated inhibition.

Binding of DKK1-AP to LRP5 and LRP5 Mutants

HEK cells were transfected with Mesd plasmids and LRP5 plasmids, asindicated in FIG. 6C, and incubated on ice with CM prepared from HEKcells expressing Dkk1-AP. The AP activity was determined in ArbitraryUnits, as described in the Materials and Methods. The expression of Wtand mutant LRP5 molecules are shown in the right panel of FIG. 6C. Theseresults indicate that LRP5R34 contains Dkk1 binding sites, and that E721in R34 is required for Dkk1 binding. (FIG. 6C).

Example 4.2 Identification of the Amino Acid Residues on the InteractionSurface on Domain III which are Required for Dkk Inhibition

As deletion of the entire third YWTD repeat domain may cause grossconformational changes in LRP5, as will be described herein, pointmutations in this domain were created that could disrupt Dkk 1-mediatedinhibition.

Schematic Representation of Ala Substitution Mutations on InteractionSurface III

The space filled model of Domain III was deduced based on the structureof the LDL receptor YWTD repeat domain (13). The homology model ofDomain III of Dkk1 was built with ICM (Molsoft L.L.C., La Jolla, Calif.)using sequences obtained from the Swiss-Prot/TrEMBL database (Entry NameQ9UP66 [18]). The Low-Density Lipoprotein (LDL) receptor YWTD-EGF domain(PDB code 1IJQ (22)) was chosen as the template. Based on thethree-dimensional structure, 19 LRP5 mutants were generated containingAla substitution mutations on the surface of Domain 111 (FIG. 7A). Theability of these mutant LRP5 proteins to resist Dkk1-mediated inhibitionwas determined and is shown in FIG. 7A. Nine of the mutants showedaltered (more than 5%) sensitivity to Dkk1-mediated inhibition, andcontained mutations localized on the same surface (FIG. 7A).

Effect of Representative Point Mutations on the Wnt Coreceptor Activityof LRP5

HEK cells were transfected with LEF activity reporter plasmids, Kremen1plasmids and expression plasmids, as indicated in FIG. 7B. Theexpression of Wt and mutant LRP5 molecules are shown in the lower panel.Among 19 mutations, the E721 mutation showed the strongest effect onDkk1-mediated inhibition, followed by W781, and Y719 (FIG. 7B).

Mutations of E721-corresponding residues in the first and second YWTDrepeat domains (D111 and D418, respectively) did not significantly alterthe sensitivity to Dkk-mediated inhibition. All the mutants that wereresistant to Dkk1-mediated inhibition were also resistant toDkk2-mediated inhibition. All this data supports the conclusion that thethird YWTD repeat domain is required for Dkk-mediated inhibition.

An obvious explanation for the requirement of the third YWTD repeatdomain for Dkk-mediated inhibition is that this domain is responsiblefor Dkk1 binding. The direct binding of Dkk1-AP fusion protein to LRP5expressed on the surface of HEK cells was measured (23).

Example 5 Screening Compounds that Interact with the Specified DomainIII of LRP5 Example 5.1 (A) Screening Compounds Using Domain III as aTemplate

Virtual Screening

The UNITY™ program (Tripos, Inc.) was used to screen the National CancerInstitute (NCI) database (http://129.43.27.140/ncidb2) for chemicalcompounds that were able to fit into the cavity on Domain III. Thisdatabase is freely searchable and includes the coordinates of 250,251small chemical compounds. A search query was designed to consist of R764and E721 with 0.3 Å tolerance, and a hydrophobic center with 1.0 Åtolerance that is 3.2 Å away from Trp781, pointing towards the cavity.Taking the flexibility of the compounds into consideration, the DirectedTweak algorithm in the UNITY™ program allowing for a rapid,conformationally flexible three dimensional search (21) was applied.

The candidate compounds obtained using the UNITY™ program were thendocked into the Dkk1 binding surface using the FlexX™ program (Tripos,Inc.) for energy minimization (17), which quickly and flexibly docksligands to protein-binding sites (44). Residues E721, W864, Y719, R764,D877, F888, G782, W781 and M891, shown to be critical for Dkk1recognition (FIG. 7A), were considered in the calculations. Followingthe docking procedures, the compounds were then ranked based on theirpredicted ability to bind to the Dkk1 binding pocket using the Cscore™program. Cscore™ generated a relative consensus score based on how wellthe individual scoring functions of the protein-ligand complexesperformed (8). The Cscore™ were then subjected to final manual visualinspection. While 40 compounds with the highest consensus scores wererequested from NCI, only 17 were obtained due to unavailability. Thesecompounds were then subjected to the Dkk-1 binding assay. Three of thesecompounds were found to have an effect on the binding of Dkk1 to LRP-5:NCI106164 (also referred to as IC14) (FIG. 8A) inhibited Dkk1 binding by32%, while NCI39914 (also referred to as IC13) (FIG. 8B) and NCI660224(also referred to as IC5) (FIG. 8C) stimulated Dkk1 binding by 645% and275%, respectively. The stimulatory effect of NCI39914 and NCI660224 maybe due to the enhanced interaction of these compounds with the Dkk1binding cavity of the third domain. This enhancement could result frombridging of the gap that exists between the interaction surfaces of Dkk1and LRP5. Since anthra-9,10-quinone (FIG. 9A) is a common substructureamong compounds NCI39914 (IC13) and NCI660224 (IC5), anthra-9,10-quinonemay play a key role in the binding interaction with LRP5. A twodimensional search for compounds found in the NCI database that aresimilar to anthra-9,10-quinone was performed using the similarity searchalgorithm of the UNITY™ program. The hits were then docked with theFlexX™ program, as previously described. 25 compounds with the highestscores were obtained from NCI and tested. Compounds NCI657566 (FIG. 9B)and NCI366218 (FIG. 10A) were able to reverse the Dkk1-mediatedinhibition of Wnt signaling. A new two dimensional similarity search wasconducted using a NCI366218-derived template shown in FIGS. 9C and 13candidate compounds were identified. Biological assays (as describedbelow) showed that NCI 8642 (FIG. 10B) was the best compound for thereversal of Dkk-mediated inhibition of Wnt signaling and the disruptionof Dkk1 binding to LRP5.

Biological Assays

Biological assays were used to screen the compounds identified byvirtual screening.

Dkk-1 Binding Assay

The binding of Dkk1-AP to HEK cells expressing full length LRP5 orLRP5R34 mutant lacking the first two domains was performed as describedin EXAMPLE 2 (FIG. 4). The first batch of 17 compounds was initiallyscreened for the inhibition of Dkk1 binding to full length LRP5. Itshould be noted that NCI106164 (IC14) showed a 68% inhibitory effect onDkk1 binding, while NCI39914 (IC5) and NCI660224 (IC3) stimulated Dkk1binding by approximately 654% and 276%, respectively (see Table I). Asfor the enhancement of the binding by IC5 and IC13, the effect may bethe result of receptor oligomerization or allosteric effects exerted onthe receptors by these symmetric molecules. Importantly, IC5, IC13 (FIG.8B), and IC15 (FIG. 8C) all contain an anthra-9,10-quinone corestructure, suggesting that the quinone structure may provide basicinteracting forces for the molecules to interact with the cavity. Thepresence of several aromatic amino acid residues in the Dkk-interactingcavity supports this idea.

TABLE I Effects of chemical compounds (2 mg/ml) on DKK1 binding BindingInhibitory Rate of DKK1 % Compound LRP5 1000 ul DMSO WT DMSO 1:100 100270071 IC1 97 45123 IC2 117 37815 IC3 85 382917 IC4 108 660224 IC5 27638290 IC6 101 649827 IC7 88 70694 IC8 180 648597 IC9 79 618567 IC10 96657726 IC11 90 12156 IC12 127 39914 IC13 654 106164 IC14 68 16221 IC1573 651656 IC16 96 67653 IC17 107

TABLE II Wnt activity assay screening of Batch II Compound Basal WntWnt + Dkk Control 100 1000 100 127133 97 170 106 1743 113 670 229 39963115 970 114 337836 116 870 81 37608 26 0 10 372294 95 0 13 123823 79 220137 366218 117 1220 476 342051 107 50 152 39957 103 40 16 4997 114 990113 116405 88 230 23 641424 111 190 19 373532 99 110 27 25869 105 880176 310659 128 130 21 28561 90 630 110 51530 130 0 0 128436 166 0 0209942 100 750 136 366105 107 100 0 159858 121 80 147 106164 88 350 64647082 95 940 105 657566 105 1140 227

TABLE III Wnt activity assay screening of Batch III Compound Basal WntWnt + Dkk Control 100 1000 240 37089 102 1090 230 97309 90 430 105 8642101 1220 1020 66425 85 1010 250 113914 92 1180 360 364163 0 0 0 11593488 800 190 110317 90 1110 250 3751 97 1090 304 28627 107 800 403 1057387 710 245 620055 10 1 6 37179 92 960 240Table II & Table III

NIH3T3 cells were transfected with Wnt activity luciferase reportergene. The next day, the compounds were dissolved in DMSO at 2 mg/ml anddiluted at 20 ug/ml into tissue culture medium (Basal), mediumcontaining Wnt3a (Wnt) or medium containing both Wnt3a and Dkk1(Wnt+Dkk). DMSO without any compound served as the control. Six hourslater, the cells were lysed and Wnt activity was determined using aluciferase assay. The data shown is percent basal activity. Compoundsthat show more than a 100% reversal of Dkk inhibition without affectingWnt activity are shown in red.

Wnt Activity Assay

The second and third domains of LRP5 are required for Wnt signaling, andthese domains probably directly interact with Wnt molecules. Since thesedomains share extensive amino acid sequence homology, it is probablethat certain compounds that bind to the third domain may also bind tothe first two domains, potentially causing the inhibition of Wntactivity. The second batch of compounds were initially screened usingthe Wnt activity assay and subsequently screened using the binding assayto confirm that compounds reversing Dkk inhibition inhibited Dkk bindingto LRP5. As shown in Table II, 25 compounds from the second batch werescreened using the Wnt activity assay. Specifically, NIH 3T3 cells weretransfected with Wnt activity luciferase reporter gene. The next day,the compounds were dissolved in DMSO at 2 mg/ml and diluted at 20 ug/mlinto tissue culture medium (B basal) containing Wn3a (Wnt) or mediumcontaining both Wnt3a and Dkk1 (Wnt+Dkk). DMSO without any compoundserved as the control. Six hours later, the cells were lysed and Wntactivity was determined using a luciferase assay. The data shown ispercent basal activity. The compounds were examined for thefollowing: 1) basal reporter activity inhibition; 2) Wnt activityinhibition; and 3) reversal of Dkk-mediated inhibition of Wnt activity.As shown in Table II, 17 out of 25 compounds were found to inhibit Wntactivity by more than 30%. Two compounds, NCI366218 and NCI657566, werefound to reverse Dkk1 mediated inhibition of Wnt signaling withoutaffecting Wnt activity.

To determine which compounds reverse Dkk-mediated inhibition, a thirdbatch of compounds was identified using virtual screening. 13 compoundswere identified and subjected to Wnt activity screening. As shown inTable III, three compounds were found to greatly inhibit Wnt activity,and one compound (NCI8642) significantly reversed Dkk-mediatedinhibition.

Both NCI8642 and NCI366218 were further characterized by Wnt activityassays and Dkk binding assays, as shown in FIG. 11 and FIG. 12. NCI8642was more effective in the reversal of Dick-mediated inhibition. NCI8642also had wider range of effective concentrations than NCI366218. Bothcompounds began to show Wnt inhibition at high concentrations. Bothcompounds reversed Dkk-mediated inhibition by disrupting the interactionbetween Dkk1 and LRP5 since both compounds inhibited the binding ofDkk1-AP to full length LRP5 and the LRP5 R34 mutant that lacks the firsttwo domains. NCI8642 was shown to be more effective than NCI366218 inthe inhibition of Dkk1 binding, consistent with its increasedeffectiveness in the reversal of Dkk-mediated antagonism to Wntsignaling.

Osteogenic Assay

Wnt stimulates the proliferation and differentiation of culturedosteoblasts and Dkk inhibits this process. Therefore, these compoundsincrease osteogenesis. This may be monitored by the examination ofmineralization or the expression of osteogeneic markers, including theexpression of BSP, osteocalcin, and collagen. The expression of GFPdriven by the 2.3 Kb Col1A1 promoter has also been monitored. FIG. 13shows that NCI366218 stimulates GFP expression suggesting an increase inosteoblast differentiation. FIG. 14 shows that NCI366218 stimulatesmineralization. NCI366218 also stimulates bone formation in calvarialorgan culture.

Example 5.1 (B) Screening Compounds Using Domain III as a Template

Additional virtual screens of the National Cancer Institute (NCI)database (http://129.43.27.140/ncidb2) were carried out for chemicalcompounds that could potentially bind to this cavity. This databaseincludes the coordinates of 250,251 small (M.W.<1,000 Da) chemicalcompounds. The initial screen was carried out with the program UNITY(Tripos, Inc.) using a search query consisting of LRP5 residues Glu721and Arg764 with 0.3 Å tolerance and a hydrophobic center with 1.0 Åtolerance that is 3.2 Å away from LRP5 residue Trp780 (69) pointingtowards the cavity (FIG. 17A). In order to take the flexibility of thecompounds into consideration, the Directed Tweak algorithm in theprogram UNITY, which allows a rapid and conformationally flexible 3Dsearch (21), was applied.

The candidate compounds (˜2000) from the UNITY screen were then dockedinto the DKK1-binding surface using the program FlexX (Tripos, Inc.),which can quickly and flexibly dock ligands to protein-binding sites(72, 73). LRP5 residues Glu721, Trp863, Tyr719, Arg764, Asp887, Phe888,Gly781, Trp780 and Met890, which have been shown to be involved in DKK1binding (69), were included in the calculations. Following the dockingprocedures, the compounds were ranked based on their predicted bindingaffinities for the Dkk1-binding pocket using the program Cscore. Cscoregenerates a relative, consensus score based on how well the individualscoring functions of the protein-ligand complex perform. The resultsfrom Cscore were then subjected to a final visual inspection.

Seventeen compounds with the highest consensus scores were furthertested (Compound IC1-IC17, Table IV). These compounds were subjected tothe Dkk-1-binding competition and Wnt activity assays. Specifically,NIH3T3 cells were transfected with Wnt activity luciferase reportergene. Next day, compounds, which were dissolved in DMSO at 2 mg/ml anddiluted into PBS at 20 ug/ml, were added into tissue culture medium(basal), medium containing Wnt3a (Wnt) or both Wnt3a and Dkk1 (Wnt+Dkk).Control contains the same amount of DMSO. Six hours late cells werelysed and Wnt activity was determined by luciferase assay. Ten compoundsshowed more than 50% inhibition of Wnt-stimulated reporter gene activitywithout significantly affecting the basal reporter gene activity (TableIV). Among these ten compounds, four affected the binding ofDkk-alkaline phosphatase (AP) to LRP5 expressed on cell surface; IC14and IC15 showed more than thirty percent inhibition, whereas,surprisingly, IC5 and IC13 showed stimulation of the binding (Table IV).The inhibition of Wnt activity may be due to the binding of thecompounds to the first two YWTD repeat domains that have been implicatedin Wnt binding probably through a direct interaction with Wnt molecules(35, 36, 47). Importantly, IC5, IC13 (FIG. 17B), and IC15 (FIG. 17C) allcontain an anthra-9,10-quinone core structure, suggesting that thequinone structure may provide basic interacting forces for the moleculesto interact with the cavity. The presence of several aromatic amino acidresidues in the Dkk-interacting cavity supports this idea.

TABLE IV First round screen summary Lef activity % Binding Com- Wnt3aCMto pound Designate Basal Wnt3aCM Dkk1CM LRP5 Structure Control Ctr 1001627 290 100 270071 IC1 104 841 314 97

45123 IC2 106 1092 318 117

37815 IC3 47 55 49 85

382917 IC4 104 452 162 108

660224 IC5 84 131 142 276

38290 IC6 114 990 323 101

649827 IC7 105 1072 370 88

70694 IC8 107 623 231 120

648597 IC9 95 268 147 79

618567 IC10 102 962 324 96

657726 IC11 109 239 217 90

12156 IC12 112 762 239 127

39914 IC13 113 147 143 654

106164 IC14 97 340 217 68

16221 IC15 133 115 106 63

651656 IC16 99 398 226 96

67653 IC17 103 1124 369 107

Control Ctr 100 1890 193 127133 IIC1 97 397 197 106

1743 IIC2 104 1420 417 81

337836 IIC4 116 1381 197 133

37608 IIC5 21 39 29 63

372294 IIC6 103 105 118 429

123823 IIC7 79 394 217 122

342051 IIC9 115 178 239 53

116405 IIC12 88 429 110 100

373532 IIC14 99 254 138 163

25869 IIC15 105 1683 369 85

310659 IIC16 128 364 147 57

28561 IIC17 90 1323 231 323

209942 IIC20 101 1565 276 805

366105 IIC21 107 288 103 54

159858 IIC22 121 269 311 118

647082 IIC23 95 1620 302 97

657566 IIC24 105 1957 552 68

TABLE V Summary for screening based on the anthra-9,10-quninone corestructure Lef activity % Wnt3aCM Compound Designate Basal Wnt3aCM Dkk1CMStructure DMSO Ctr 100 1890 193 39963 IIC3 115 1536 229

366218 IIC8 109 2392 713

39957 IIC10 103 161 120

4997 IIC11 111 1782 223

641424 IIC13 111 443 128

51530 IIC18 129 119 127

128436 IIC19 166 144 148

TABLE VI Summary for screening based on IIC15 Lef activity % Wnt3aCMCompound Designate Basal Wnt3aCM Dkk1CM Structure DMSO 20 μg/ml 100 1070339 37089 IIIC1 102 1160 362

97309 IIIC2 90 503 193

66425 IIIC4 85 1071 358

113914 IIIC5 95 1238 463

115934 IIIC7 88 865 276

110317 IIIC8 90 1505 426

3751 IIIC9 97 1491 477

28627 IIIC10 107 1133 613

10573 IIIC11 87 999 404

37179 IIIC13 92 1323 391

TABLE VII Summary for screening based on IIC24 Lef activity % Wnt3aCMCompound Designate Basal Wnt3aCM Dkk1CM Structure DMSO 20 μg/ml 100 1070339 8642 IIIC3 101 1282 1111

364163 IIIC6 63 53 42

620055 IIIC12 10 12 15

IIC8 and IIIC3 are in blue. Color legends are the same as inSupplementary Table I.

With the assumption that the quinone core structure may contribute tothe interaction, we conducted a second round of virtual screening and a2D search, for the compounds that are similar to anthra-9,10-quinone inthe NCI database using the similarity search algorithm of the UNITYprogram. The hits were then docked with FlexX as described previously.Seven highly scored compounds were then obtained from NCI and tested inthe biological assays. Compound IIC8 (FIG. 17D) was identified for itsability to reverse Dkk-mediated inhibition of Wnt signaling (Table V).

In addition to the anthra-9,10-quinone-based compounds, an additional 17compounds that ranked from 41-80 in their Cscores from the first virtualscreen was obtained. The test of these 17 compounds revealed that nineof them inhibited Wnt signaling more than 50% (shaded compounds in TableIV). Importantly, four of compounds from this screen, including IIC15(FIG. 17E) and IIC24 (FIG. 17F), showed some ability to reverseDkk-mediated inhibition of Wnt signaling without significantlyinhibiting Wnt signaling itself (Table IV). A 2D similarity search wascarried out using IIC15 and IIC24 as the templates, and 13 additionalcandidate compounds were obtained for the Wnt activity assay (Tables VIand VII). One of the IIC24-based compounds, IIIC3 (FIG. 17G), turned outto be a very potent antagonist of Dkk-mediated inhibition of Wntsignaling (Table VII).

Both IIC8 and IIIC3 compounds were characterized further in the Wntactivity and Dkk binding assays. IIIC3 is more effective in reversal ofDkk inhibition and has a wider range of effective concentrations thanIIC8 (FIG. 18A, B). IIC8 started to reverse Dkk-mediated inhibition atmicro molar concentrations with an approximate EC₅₀ of 10 μM, whereasIIIC3 started to reverse Dkk inhibition below 1 μM with an EC50 of about2 μM. Both compounds appeared to inhibit Wnt signaling at highconcentrations. This inhibitory effect may be due to the low affinity ofthe compounds for the first two YWTD repeat domains that are requiredfor Wnt signaling. Because both compounds inhibited the binding ofDkk1-AP to wildtype LRP5 (FIG. 18C) and a LRP5 mutant (FIG. 18D) thatlacks the first two YWTD repeat domains, but retaining the Dkk bindingdomain (69), it is reasonable to conclude that these compounds are mostlikely to reverse Dkk inhibition by disrupting the interaction betweenDkk1 and LRP. In addition, IIIC3 is more effective than IIC8 in theinhibition of Dkk1 binding, which is consistent with its increasedeffectiveness in its reversal of Dkk-mediated antagonism to Wntsignaling. Furthermore, the IC₅₀ values of these compounds (about 1 μMfor IIIC3 and 10 μM for IIC8) for blocking the binding of Dkk1 to LRP5are similar to the EC₅₀ values for these compounds to reverse Dkkinhibition, further supporting the conclusion that these compoundsreverse Dkk inhibition by disrupting the interaction between Dkk andLRP.

To better understand the function and structure relationship of thecompounds, Wnt inhibiting compounds were reexamined. Among the 54compounds we tested, almost a half (74) of them showed more than 50%inhibition of Wnt3a at 20 μg/ml (Tables IV-VII). The most potent Wntinhibitor is IC15 (FIG. 17C). IC15 has an IC₅₀ value of about 0.5 μM forinhibiting the Wnt3a activity (FIG. 19A). This value is about 10 foldlower than the IC₅₀ value (5 μM) for inhibiting Dkk binding to the thirdYWTD EGF repeat domain, the domain that is required for Dkk antagonism(FIG. 19A). By contrast, the concentrations of IIIC3 required forinhibiting Wnt are at least ten times higher than those required forinhibiting Dkk binding, while the IC₅₀ values for IIIC3 to inhibit Dkkbinding and to reverse Dkk-inhibition are similar (FIG. 18). Theseconclusions are further supported by the molecular modeling analysis ofthe binding of IIIC3 and IC15 to the second and third YWTD repeatdomains. Although the second and third YWTD repeat domains of LRP5 shareextensive amino acid sequence homology (41% identity), there areconsiderable differences in the ligand binding cavities of these twodomains (FIG. 29). The cavity on the second domain is larger than thaton the third the domain, while the cavity of the third domain is deeper(FIG. 19 B, C). Therefore, it is reasonable to predict that IC15, whichis larger than IIIC3, may fit better into the cavity of the seconddomain, whereas IIIC3 may match better with the cavity of the thirddomain. Indeed, in the FlexX docking models, the IIIC3 fits well intothe cavity of the third domain (FIG. 3B), whereas IC15 fits perfectlywith that of the second domain (FIG. 19C). In the IIIC3 complexstructure, if the third domain is replaced with the second domain, thebound IIIC3 will lose many contacts with the target residues, thatinclude Glu721, Tyr719, Leu823, and Phe888 (FIG. 19D). On the otherhand, if the second domain is replaced with the third domain in the IC15complex, the residues, for instance Leu823 and Phe888, on the thirddomain would clash with the bound IC15 (FIG. 19E).

It was next determined whether the most potent Dkk antagonistic compoundIIIC3 could stimulate bone formation in mice. The compound was tested ina local bone formation model. IIIC3 was dissolved in DMSO and diluted1:1000 into PBS at a concentration of 0.44 mg/ml. Fifteen micro-litersof IIIC3 (0.22 mg/Kg/day), control vehicle, or positive control (b-FGF,12.5 μg/Kg/day) were injected into the subcutaneous tissue over theright side of the calvaria of four weeks old CD-1 mice three times a dayfor 5 days using a injection method described previously (74, 75).Calvarias were collected 22 days after the first injection and fixed forsectioning. As shown in FIG. 19, new bones were found in both b-FGF andIIIC3 treated calvarias. IIIC3 appeared to be as potent as FGF instimulating bone formation (FIG. 19B). To evaluate the effectiveness ofthe compound in a systemic bone formation assay, we injected C57B1 mice(8 weeks old) intraperitoneally with IIIC3 and control vehicle. Weobserved significant increases in both femoral and whole body bonemineral density in mice injected with IIIC3 (2% increase for femur and2.5% for total) compared to the control (FIG. 19 C, D), accompanyingwith a significant increase in body weight (7.1%; FIG. 19E). Theseresults further validated the LRP/Dkk interaction as a potentialtherapeutic target for increasing bone formation and demonstrated theseDkk antagonists may be suitable lead compounds for further developmentinto anabolic therapeutics to treat osteoporosis.

Protein-protein interactions are generally considered to be difficult todisrupt by small molecule compounds. In this study, a screening approachwas used that combines structural biology, computation, and biologicalassays to identify small molecule compounds that can efficiently disruptDkk-LRP5 interaction. A de novo initial virtual screen was carried outfor chemicals based only on a predicted structure of a target withoutany template. We were able to identify compounds from physical screeningof merely 54 compounds, and the successive rounds of screening yieldedbetter compounds than the previous did. In addition, the computationallows not only simply identifying compounds that fit into a cavity, butalso provides a guide to compounds that can discriminate two similarcavities, i.e., the third YWTD repeat domain from the first two YWTDrepeat domains of LRP5. The approach has allowed us to identify potentDkk and Wnt antagonistic compounds that have shown great potentials tobe developed into therapeutic reagents for treating osteoporosis andcancer. In addition, these compounds provide useful tools fortherapeutic target validation and basic research in Wnt signaling.

Example 6 Use of IIIC3 as a Core Compound to Design New Variants

As described above, the ability of IIIC3 (NCI 8642) to act upon Wntactivity allows it to be used to design a core model where varying the Rgroups on this template allows identification of other molecules thatmay also have effects upon Wnt activity. Thus, if like IIIC3, R¹, R³, R⁴and R⁶ were hydrogens and R⁸ was a Hydroxyl group, a more limited seriesof compounds could be made with the remaining positions of the followingmodel compound (VIII):

To initiate this series, a panel of compounds have been designed whereR¹¹ and R¹² are methyl groups and R¹³ is a hydroxyl group as in IIIC3and the amine group was quarternized, giving the structure (VI):

A series of substitutions have been designed for variations of R¹⁵ inthis compound using both linear and branched alkanes. The structures ofthe resultant compounds (EnzoM01-EnzoM12) are scanned as describedpreviously to obtain a score for their likelihood of binding. A list ofthe particular substitutions used in EnzoM01-EnzoM12 as well as theresultant Cscore ratings are given below:

TABLE VIII Compound R¹⁵ Cscore IIIC3 — 4 EnzoM01 CH₃ 5 EnzoM02 CH₂CH₃ 5EnzoM03 (CH₂)₂CH₃ 5 EnzoM04 CH(CH₃)₂ 4 EnzoM05 (CH₂)₃CH₃ 3 EnzoM06CH₂CH(CH₃)₂ 5 EnzoM07 CH(CH₃)(CH₂CH₃) 4 EnzoM08 C(CH₃)₃ 4 EnzoM09(CH₂)₄CH₃ 4 EnzoM10 (CH₂)₂CH(CH₃)₂ 4 EnzoM11 CH₂CH(CH₃)(CH₂CH₃) 5EnzoM12 CH₂C(CH₃)₃ 5

In another approach, the carboxyl group of NCI 8642 is replaced by acarboxamide group to generate a series of compounds with the generalstructure (VII):

A list of the particular substitutions used in this series(EnzoM13-EnzoM41) as well as the resultant scores are given below:

TABLE IX Compound R¹³ R¹⁴ Cscore EnzoM13 H H 5 EnzoM14 H CH₃ 5 EnzoM15CH₃ CH₃ 5 EnzoM16 H CH₂CH₃ 5 EnzoM17 H (CH₂)₂CH₃ 5 EnzoM18 CH₃ CH₂CH₃ 4EnzoM19 CH₃ (CH₂)₂CH₃ 5 EnzoM20 H C(CH₃)₃ 5 EnzoM21 H (CH₂)₃CH₃ 5EnzoM22 CH₃ (CH₂)₃CH₃ 5 EnzoM23 H CH₂CH(CH₃) 5 EnzoM24 CH₃ CH₂CH(CH₃)₂ 5EnzoM25 CH₃ C(CH₃)₃ 5 EnzoM26 CH₂CH₃ (CH₂)₂CH₃ 5 EnzoM27 CH₂CH₃ CH(CH₃)₂5 EnzoM28 CH₂CH₃ (CH₂)₃CH₃ 5 EnzoM29 CH₂CH₃ CH₂CH(CH₃)₂ 3 EnzoM30 CH₂CH₃(CH₂)₃CH₃ 5 EnzoM31 CH₂CH₃ CH₂CH(CH₃)₂ 3 EnzoM32 CH₃ (CH₂)₄CH₃ 5 EnzoM33CH₃ (CH₂)₂CH(CH₃)₂ 5 EnzoM34 H (CH₂)₄CH₃ 5 EnzoM35 H (CH₂)₂CH(CH₃)₂ 5EnzoM36 CH₂CH₃ (CH₂)₄CH₃ 5 EnzoM37 CH₂CH₃ (CH₂)₂CH(CH₃)₂ 5 EnzoM38CH₂CH₃ CH₂CH(CH₃)(CH₂CH₃) 2 EnzoM39 H CH₂CH(CH₃)(CH₂CH₃) 5 EnzoM40 HCH₂C(CH₃)₃ 2 EnzoM41 CH₂CH₃ CH₂C(CH₃)₃ 4

In another series of compounds, the carboxyl group is esterified to givethe structure (VIII):

A panel of compounds (EnzoM42-EnzoM70) were designed with variousgroups; these substitutions and cScores are given below:

TABLE X Compound R¹³ Cscore EnzoM42 CH₃ 3 EnzoM43 CH₂CH₃ 5 EnzoM44(CH₂)₂CH₃ 5 EnzoM45 CH(CH₃)₂ 5 EnzoM46 (CH₂)₃CH₃ 5 EnzoM47 CH₂CH(CH₃)₂ 5EnzoM48 CH(CH₃)(CH₂CH₃) 5 EnzoM49 C(CH₃)₃ 5 EnzoM50

5 EnzoM51

5 EnzoM52

5 EnzoM53

2 EnzoM54

5 EnzoM55

2 EnzoM56

4 EnzoM57

3 EnzoM58

2 EnzoM59

4 EnzoM60

5 EnzoM61

2 EnzoM62

2 EnzoM64

2 EnzoM65

2 EnzoM66 (CH₂)₄CH₃ 5 EnzoM67 (CH₂)₂CH(CH₃)₂ 5 EnzoM68CH₃CH(CH₃)(CH₂CH₃) 5 EnzoM70 CH₂C(CH₃)₃ 4

It can be seen that the variety of substitutions that have been made injust three sites on the core molecule were able to generate a largenumber of candidates that can be tested by virtual screening withoutsynthesizing a single molecule. Furthermore, when this series ofcompounds were tested in the same virtual screening program describedpreviously, 44 out of the 70 compounds gave cScore values of 5. Thisdemonstrates the power of the virtual substitution technique indesigning new compounds since the compound IIIC3 used to design thesemolecules only had a relative cScore rating of 4.

Example 7 Refinement of Scores

Although all of the compounds that achieved high cScores in the previousexample are candidates for testing, it would be more efficient tosynthesize and test the ones that are the most likely to be effective.Since there was so many that had ratings of 5 (the highest potentialcScore), a different criterion has to be used to sort the compounds out.In computing the cScore, various component scores are compiled togetherto achieve a final rating. In the original screening of the NCI library,the template compound, NCI 18642 (IIIC3), showed the highest rating ofone of these components, the FR-Score. As such, this component score wasused to rank the various analogs of the core compound that had cScoresof 5. When viewed this way, 7 of the 44 candidates scored higher thanthe original NCI 18642. The values for these compounds (as well as thosefor IIIC3) are given below in Table XI.

TABLE XI Com- FR- GR- PMFR- DR- pound Score Score Score Score ChemscorecScore EnzoM14 −17.76 −156.56 −74.49 −94.41 −27.04 5 EnzoM15 −16.59−130.66 −72.09 −94.78 −27.46 5 EnzoM25 −16.31 −162.33 −72.71 −108.31−30.04 5 EnzoM12 −16.19 −183.24 −107.71 −103.93 −28.91 5 EnzoM01 −15.57158.54 −93.24 −91.66 −27.32 5 EnzoM39 −15.57 182.99 −82.66 −106.61−27.99 5 EnzoM35 −15.23 172.59 −84.19 −104.16 −28.42 5 IIIC3 −14.79−124.58 −71.08 −73.64 −26.28 4

This approach again shows the ability of screening a virtual library toidentify promising candidates that would be worth synthesizing andtesting in biological assays. Due to their higher rankings, it ispossible that such candidates may demonstrate more potency than theoriginal compounds that defined a core structure.

Example 8 Synthesis of Compounds Selected from Example 7

Synthesis of compounds selected by the virtual screening steps describedabove can be carried out by a number of means known in the art. Forinstance, these compounds may be created by taking a similar compoundand substituting chemical groups by one or more replacement steps. In anexample of this, IIIC3 (NCI 18642) is commercially available asgallocyanine and can serve as a starting point for many substitutions.Alternatively, precursors of the desired compound can be synthesizedwith the appropriate R groups and joined together to synthesize acandidate to be tested in a biological assay.

(A) Synthesis of EnzoM15

To a solution of 6.02 g of gallocyanine (free of hydrochloride) in 40 mlof DMF was added 6.62 g of succinimido-1,1,3,3-tetramethyluroniumtetrafluoroborate followed by 6.96 ml of diisopropylethylamine andstirred overnight at room temperature. After removal of DMF, the residuewas resuspended in 10 ml of anhydrous THF and then 10 ml of 2Mdimethylamine in TMF was added. The mixture was heated at 50-60° C. for5 hours. The product was purified by column chromatography using silicagel (CH₂Cl₂/1% MeOH) to give 0.8 g (12% yield) of EnzoM15 (shown below).

(B) Synthesis of EnzoM14

The synthesis of EnzoM14 was carried out basically as described forEnzoM15 except that 100 ml of methylamine was substituted for thedimethylamine used in the synthesis of EnzoM15. The heating step wasalso increased from 5 hours to overnight instead. Purification on silicagel was carried out as described above to give 0.75 g (12% yield) ofEnzoM14 (shown below).

(C) Synthesis of EnzoM01Step 1. Synthesis of Methyl Iodide

A three necked flask with a stirring magnet was fitted with athermometer, a separatory funnel and a small fractionating columnconnected with a condenser set for downward distillation and a receivingflask kept in an ice water bath. A solution was made by dissolving 800 g(4.8 moles) of potassium iodide in 430 ml of water and this was added tothe flask. 60 g of calcium carbonate was then added and the mixtureheated to 60-65° C. with stirring. The temperature was maintained at60-65° C. and then 630 g (473 ml, 5 moles) of “practical” methyl sulfatewas gradually added through the separatory funnel over a period of 2hours. The rate of addition of the calcium carbonate was maintained tokeep distillation of methyl iodide product proceeding into the receivingflask. After all of the calcium carbonate had been added, thetemperature was raised to 65-70° C. for about 40 minutes to completedistillation of the methyl iodide. The product was separated from asmall amount of water residue and then the product was dried overanhydrous calcium chloride followed by decanting the methyl chlorideinto a dry distilling flask. A few crystals of potassium iodide wereadded and the product distilled from a water bath. The redistilledproduct gave a yield of 615-640 g (a 90-94% yield) and had a boilingpoint of 41-43° C.

Step 2. Addition of Methyl Group to Gallocyanine

1.5 g of gallocyanine (chloride free) was dissolved in 500 ml of DMF ina 1000 ml flask. The mixture was stirred at 30° C. for 1 hour and then10 ml of the methyl iodide made in the step above was added. Thesolution was then heated and refluxed with stirring for 72 hours, thecondenser pipe was sealed with a balloon and additional 10 ml of methyliodide was added every 12 hours. The THF was then distilled off at 80°C. The remaining solid (EnzoM01) gave a yield of about 98%.

A mixture of 100 mg (0.33 mMoles) of gallocyanine (chloride free), 50 mlof methanol, 38 μl (0.33 mMoles) of 2,6-lutidine and 0.5 ml of methyliodide were placed in a pressure vessel with stirrer. The mixture wasstirred and heated at about 100° C. for 4 days. After concentration invacuo, the residue was washed with 5 ml of ethanol. Filtration gave 91mg of solid product. The structure of EnzoM01 is given below:

(D) Synthesis of EnzoM02

Since EnzoM01 showed interesting results (results shown in Examplesbelow), a related compound, EnzoM02, that had a FR-Score lower than theoriginal IIIC3, was also chosen for synthesis and biological testing.The compound EnzoM02 is essentially similar to EnzoM01 except that anethyl group replaces one of the methyl groups on the quarternizednitrogen. As such, EnzoM02 was made essentially as described for EnzoM1except that the quarternization took place with ethyl iodide instead ofmethyl iodide and it was carried out on a smaller scale. A mixture of100 mg (0.33 mMoles) of Gallocyanine (chloride free), 50 ml of methanol,38 μl (0.33 mMoles) of 2,6-lutidine and 0.5 ml of methyl iodide wereplaced in a pressure vessel with stirrer. The mixture was stirred andheated at about 100° C. for 4 days. After concentration in vacuo, theresidue was washed with 5 ml of ethanol. The yield of filtration productwas 134 mg and the structure of EnzoM02 is shown below:

(E) Synthesis of EnzoM03

Another compound EnzoM03 that was related to EnzoM01 (but like Enzo M02in having an FR-Score lower than IIIC3) was chosen for synthesis. Thecompound EnzoM03 differs from EnzoM01 by having a propyl group insteadof a methyl group on the quarternized nitrogen. As such, the compoundEnzoM03 was made essentially as described for EnzoM02 except that thequarternization took place with propyl iodide instead of ethyl iodide.The yield of filtration product was 138 mg and the structure of EnzoM03is shown below:

Example 9 Testing Compounds Synthesized in Example 8

After synthesis of the compounds described in Example 8, they weretested as described previously for Wnt activity and effects on Dkkinhibition. Additionally, the previous screening had used a singleconcentration of the test compound in the assays. Since a smaller numberof compounds were being tested in this example, a variety of differentinputs for each test compound could be tested simultaneously for Wntactivity. The results of various concentrations of EnzoM01 are shown inFIG. 21 and as a comparison, a titration with IC15 is also included. Itcan be seen for both EnzoM01 and IC15, there is a dose response foreffects upon Wnt activity. However, the same level of inhibition of Wntactivity seen by IC15 can be seen at a dose of EnzoM01 that isapproximately 15 times less. It can also be seen that at higher levelsof EnzoM01 (8.33 μM and greater) there is a change in the effect uponWnt activity, possibly indicating a more complex response to this drug.An HPLC analysis of this particular EnzoM01 preparation also showed itto be contaminated with a small amount of unreacted gallocyanine whichmay also be a factor at the higher dosages.

In FIG. 22, the results are shown for EnzoM14 and EnzoM15. In thisexperiment, EnzoM14 shows a dose response for reduction of Wnt activitywhereas EnzoM15 shows a more abrupt response after 6.1 μM. It can alsobe seen that these compounds have no apparent effect on Dkk-mediatedinhibition of Wnt activity.

In FIG. 23, the results are shown for EnzoM02 and Enzo M03 withessentially similar results for each compound. At the concentrationsused in this experiment there was very slight effects upon Wnt activityitself. However, at the highest concentrations used (6 μM and 30 μM),these compounds demonstrated an ability to decrease the effects of Dkkon Wnt activity.

Example 10 Effects of Various Compounds on the Viability of a Tumor CellLine

As described above, the Wnt pathway has been linked to the developmentand progression of cancer. Three of the compounds that have beendiscovered to affect Wnt activity (IC15, IIIC3 and EnzoM01) were appliedto a tumor cell line (PC-3) growing in vitro.

PC-3 tumor cells were seeded into a 96 well plate with: A) 500cells/well; B) 1000 cells/well; and C) 2000 cells/well with Ham's F12medium supplemented with 10% FBS. Various amounts of IC15, IIIC3 orEnzoM01 (10 μM. 20 μM or 80 μM final) were added to the media and growthcontinued for 10 days. Media was removed, the cells were washed with PBSand then incubated with RPMI media (without Phenol Red) containing 20%Cell titer 96 Aqueous One Solution Reagent (Promega, Madison, Wis.) forfour hours. Cellular proliferation was then measured by absorbanceaccording to the manufacturer's directions. The results of this assayare shown in FIG. 24. The presence of these compounds seemed to have aprofound effect upon the number of viable PC-3 cells present at the endof the experiment, however, the effects of the drug was dependent uponthe initial plating density. At the lowest density (500 cells/well)IIIC3 and EnzoM01 showed a decrease in the number of viable cells at 10μM and IC15 showed an effect at 20 μM. In the 1000 cell/well samples,IIIC3 and EnzoM01 show a 10% and 15% reduction in the number of viablecells at the 10 μM level, respectively, and approximately a 50%reduction at the 20 μM level; whereas, IC15 shows a reduction only atthe highest concentration tested, 80 μM. At the highest cell densitytested (2000 cells/well) all three compounds demonstrated cytotoxicityat levels greater than 20 μM of test compounds were present. Inconclusion, this example demonstrates that three different compoundsselected for an ability to a) bind to LRP 5/6 and b) affect Wnt activityare capable of reducing the ability of a cancer cell line toproliferate.

Example 11 Effects on β-catenin Activity

The protein β-catenin is considered to be a major downstream target ofthe Wnt pathway. It had previously been noted that a number of celllines derived from tumors have elevated levels of β-catenin compared tonormal cells. In fact, various mutations and deletions in the apc(adenomatous polyposis coli) gene, which directly regulates β-cateninactivity, has been strongly linked with the development of colon cancer.For instance, mice strains that have been genetically engineered to havedefects in the apc gene (“multiple intestinal neoplasia” or “min”strains) are genetically predisposed to develop a large number ofintestinal tumors as they age.

A number of different tumor cell lines were tested for the effects ofEnzoM01 on expression of β-catenin as measured by an ELISA assay. Thetumor lines were derived from different tissue types: ovarian cancer(PA-1), prostate cancer (PC-3), breast cancer (HTB-24 and HTB26) andcolon cancer (Lys174T).

A) Cell Growth

The various cell lines listed above were grown to about 70% confluencyand then various amounts of EnzoM01 were added and incubated for 16hours. The media was removed, the cells harvested and cellular lysateswere used for ELISA assays to measure (β-catenin.

B) ELISA Assay

Wells of a 96 well plate were coated with capture antibody, ananti-β-catenin monoclonal antibody (BD Bioscience, San Diego Calif.), insodium carbonate, pH 9.0 overnight at 4° C. The next day, the wells werewashed once with TBST (Tris buffered saline containing 0.02% Tween 20)and incubated with 1% BSA blocking solution for 1 hour at roomtemperature. Wells were washed once with TBST and cell lysates added andincubated at room temperature for 1 hour. Wells were then incubated withanti-β-catenin polyclonal antibody (BD Bioscience, San Diego, Calif.) atroom temperature for 45 minutes. Wells were washed three times with TBSTand then a peroxidase conjugated antiRabbit IgG secondary antibody (CellSignaling Technology, Danvers, Mass.) was added to each well andincubated for 25 minutes. Wells were washed three times with TBST andfluorescence was developed by incubating with 1:1 mix of SuperSignalWest Pico Stable Peroxide Solution (Pierce Biochemicals, Rockford,Ill.).

C) Results

The results of this experiment are shown in FIG. 25A. It can be seenthat the intrinsic level of β-catenin in LS174T is much higher comparedto the other cell lines and that there is a slight but dose-responsiveeffect upon LS174T by EnzoM01 at the dosages used where β-cateninactivity actually increases as the dosage goes up. However, since theactivity of β-catenin is so high in this cell line, it obscures theeffects on the other cell lines that have much lower intrinsic levels ofβ-catenin activity. Accordingly, this data is also presented with theLS174T data omitted in FIG. 25B. It can be seen that although there wasessentially no change in β-catenin levels in the breast cancer celllines HTB-26 and HTB24 as well as the prostate cancer cell line PC-3,there was a selective reduction of β-catenin levels in the ovarian cellline PA1 at the higher doses of EnzoM01. The IC50 value for EnzoM01 wasin the range of 20-40 μM.

Example 12 Effects of Various Compounds on Tumor Induction andProgression

In a study on the effects of diet upon induction and progression ofcolon tumors, apc^(min/+) mice had been shown to respond to the presenceor absence of a particular dietetic supplement, β-glucosylceramide(110). The ability of a reagent to influence the frequency or size ofspontaneous tumors in this strain of mice presents an opportunity totest some of the previously identified compounds for their potential inaffecting induction and progression of cancer in a live animal.

Compounds IC15 and IIIC3 were identified in the virtual screeningprocess for a likelihood of binding to a selected site on LRP5. Both ofthese compounds were administered to groups of apc^(min/+) mice and thesize and frequency of tumors were measured at a later time point.

A) Treatment of Test Mice

In order to investigate the effectiveness of these two compounds invivo, 8-9 week old C57BL/6J^(APC(min+))/J were obtained from JacksonLaboratories (Bar Harbor, Me.). The mice were kept on a normal diet for54 days at which time they were switched to a high fat diet for 36 days.At this point, the mice were injected with vehicle, 625 uM IIIC3 or 42uM IC15 in a volume of 1 ml every other day for 90 days. At the end ofthe treatment period, the mice were sacrificed and their intestinesremoved and followed by staining with methylene blue to quantify thenumber and size of developing tumors in the intestines.

B) Results

The number of tumors of various sizes is given below in Table XII.

TABLE XII Tumors Tumors of size of size Tumors of Tumors of Tumors ofTumors of Total Treatment 0-1.53 mm² 1.53-3.0 mm² size 3.0-4.62 mm² size4.62-6.15 mm² size 6.15-7.69 mm² size >7.69 mm² Tumors Vehicle 20.7 ±2.08 18.57 ± 2.98 11.43 ± 3.44  4.71 ± 0.89  2.57 ± 0.37 1.43 ± 0.4859.43 ± 8.18 IIIC3  7.5 ± 1.38 10.33 ± 1.48 6.50 ± 0.96 2.5 ± 0.67 2.5 ±0.5 2.33 ± 0.67 31.67 ± 3.49 1C15 9.33 ± 1.33 16.00 ± 0.58 4.67 ± 1.453.3 ± 0.88  3.7 ± 1.87 1.33 ± 0.33 38.33 ± 3.71

It can be seen that both of these compounds gave a decrease in theoverall number of tumors. The average number of tumors on the testanimals was 59.4 for the control and only 31.67 and 38.33 for theanimals treated with IIIC3 and IC15 respectively. This shows a reductionof 47% and 36% after treatment with these compounds. It can also be seenthat the effects of these compounds is seen more prominently with thesmaller sized tumors than the larger tumors. This may be an indicationthat these compounds delay initiation but have less of an effect uponprogression once a tumor has been established although otherexplanations are also possible.

Example 13 Effects of Various Compounds on Levels of Glucose, Insulin,Triglycerides and Cholesterol in Mice on a High Caloric Diet

As has been discussed previously, LRP5/6 has been shown to play multipleroles in the Wnt signaling pathway, including the modulation of normalcholesterol and glucose metabolism. A group of C57BL/6J mice wereinitially maintained on a normal caloric diet for 5 days after whichtime they were fasted overnight and then blood glucose levels wereassayed using a Lifescan glucometer (Johnson & Johnson). The animalswere then placed on a high caloric diet for 10 days during which timethey were simultaneously administered different concentrations ofEnzoM01, IC15 and IIIC3 (0.02, 0.06 and 0.30 mg/kg/day for Enzo M01,0.2, 0.6 and 3.0 mg/kg/day for IC15 and 0.5, 2.0 and 8.0 mg/kg/day forIIIC3). On the final day, animals were subjected to an overnight fastand then blood glucose levels were determined.

Results

After feeding on a high caloric diet for 10 days, the control miceshowed an approximately 30% increase in serum glucose levels. In FIG. 26it can be seen that all three compounds differentially lowered bloodglucose levels to varying degrees. At the highest concentrations used,both IIIC3 and IC15 brought blood glucose levels back to within 10% ofnormal, physiological serum values; whereas, treatment with EnzoM01 wasmost effective at a concentration of 0.02 mg/kg/day. These resultsdemonstrate that all three compounds are capable of returninghyperglycemic mice back to normo-glycemic values without inducinghypoglycemia. In addition, blood samples were also analyzed forcholesterol and triglyceride levels. This was carried out by submittingserum samples to a clinical laboratory (Enzo Laboratories, Farmingdale,N.Y.) where they were measured the same way as if they were patientsamples. As seen in FIG. 27A, EnzoM01, and in 27B, IC15, were effectivein lowering triglyceride levels. At the concentrations used, it can alsobe seen in FIG. 27A that EnzoM01 was also effective in loweringcholesterol

Example 14 Treatment of Diabetic Mice

In addition to investigating the effect these compounds have on normalmice fed a high caloric diet, markedly diabetic and insulin-resistantdb/db mice (Jackson Laboratories, Bar Harbor, Me.) were tested withtreatment with IIIC3. The db/db mouse is hyperleptinemic and developsobesity and severe type 2 diabetes partly due to a functional defect inthe long-form leptin receptor, which plays a significant role in theregulation of food intake and the control of body weight (111, 112).

The diabetic prone mice were placed on a high caloric diet for 12 daysduring which time they were simultaneously administered 7 mg/kg/day ofIIIC3. At the end of this treatment, the mice were fasted for 12 hoursand the mice were then administered an IP injection of 1 g/kg glucosefor a glucose tolerance test. At various times after the injection,glucose and insulin concentrations in the blood were analyzed withglucose being measured as described previously and insulin measured bythe Insulin Ultrasensitive EIA (ALPCO Diagnostics, Salem, N.H.). As seenin FIG. 28A, the mice treated with IIIC3 demonstrated an improvedability for glucose disposal. This effect was also paralleled by asignificant reduction of plasma insulin levels (FIG. 28B).

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

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1. A compound having the structure (VI):

wherein R¹⁵ is a linear or branched alkyl group.
 2. The compound ofclaim 1, wherein R¹⁵ is a linear or branched C₁₋₅ alkyl group.
 3. Thecompound of claim 1, wherein R¹⁵ is a methyl group.
 4. The compound ofclaim 1, wherein R¹⁵ is an ethyl group.
 5. The compound of claim 1,wherein R¹⁵ is a propyl group.
 6. The compound of claim 1, wherein R¹⁵is CH₂C(CH₃)₃.
 7. A composition comprising the compound of claim
 1. 8.The compound of claim 1, wherein R¹⁵ is CH(CH₃)₂.
 9. The compound ofclaim 1, wherein R¹⁵ is (CH₂)₃CH₃.
 10. The compound of claim 1, whereinR¹⁵ is CH₂CH(CH₃)₂.
 11. The compound of claim 1, wherein R¹⁵ isCH(CH₃)(CH₂CH₃).
 12. The compound of claim 1, wherein R¹⁵ is C(CH₃)₃.13. The compound of claim 1, wherein R¹⁵ is (CH₂)₄CH₃.
 14. The compoundof claim 1, wherein R¹⁵ is (CH₂)₂CH(CH₃)₂.
 15. The compound of claim 1,wherein R¹⁵ is CH₂CH(CH₃)(CH₂CH₃).
 16. The composition of claim 7,further comprising a pharmaceutically acceptable excipient.
 17. Thecomposition of claim 16, further comprising a second compound, whereinthe second compound is a therapeutic agent.